Novel caviidae allergens and uses thereof

ABSTRACT

The present invention relates to the provision of a nucleic acid molecule encoding a Caviidae allergen comprising a polynucleotide selected from the group consisting of (a) a polynucleotide sequence as shown in SEQ ID NO 1 (Cav p 3), SEQ ID NO 5 (Cav p 2), SEQ ID NO 3 (Cav p 6) or SEQ ID NO 19 ( Cavia porcellus  allergen 1 a) or a fragment thereof, (b) a polynucleotide sequence encoding a polypeptide as shown in SEQ ID NO 2 (Cav p 3), SEQ ID NO 6 (Cav p 2), SEQ ID NO 4 (Cav p 6) or SEQ ID NO 20 ( Cavia porcellus  allergen 1 a) or a fragment thereof (c) a polynucleotide sequence which has at least 80% identity to the polynucleotides as defined in (a) or (b) encoding a Caviidae allergen or a fragment thereof, (d) a polynucleotide sequence encoding a polypeptide which has at least 85% identity to the polypeptide as shown in SEQ ID NO 2 (Cav p 3), SEQ ID NO 6 (Cav p 2), SEQ ID NO 4 (Cav p 6) or SEQ ID NO 20 ( Cavia porcellus  allergen 1 a ) or a fragment thereof, (e) a polynucleotide sequence which hybridizes to the polynucleotide sequence of any one of (a) to (d) and whereby the coding strand encodes a Caviidae allergen or a fragment thereof, (f) a polynucleotide sequence encoding a polypeptide as encoded by the nucleotide sequence of any one of (a) to (e) wherein at least one amino acid is deleted, substituted inserted or added and whereby said polynucleotide encodes a Caviidae allergen or a fragment thereof, and (g) a polynucleotide sequence being degenerate as a result of the generic code to the polynucleotide sequence as defined in any one of (a) to (f) The present invention also provides for a method for producing the polypeptides (Cav p3, Cav p6,  Cavia porcellus  allergen 1 a) encoded by said polynucleotides Moreover, the use of such allergens in a medical setting (e g in form of a pharmaceutical and/or diagnostic composition) an in vitro method for determining the allergenicity to Caviidae as well as an method for identifying potential antagonists or agonists of the polypeptides are also provided in the present invention.

The present invention relates to the provision of a nucleic acidmolecule encoding a Caviidae allergen comprising a polynucleotideselected from the group consisting of (a) a polynucleotide sequence asshown in SEQ ID NO:1 (Cav p 3), SEQ ID NO:5 (Cav p 2), SEQ ID NO:3 (Cavp 6) or SEQ ID NO: 19 (Cavia porcellus allergen 1a) or a fragmentthereof; (b) a polynucleotide sequence encoding a polypeptide as shownin SEQ ID NO:2 (Cav p 3), SEQ ID NO:6 (Cav p 2), SEQ ID NO:4 (Cav p 6)or SEQ ID NO: 20 (Cavia porcellus allergen 1a) or a fragment thereof;(c) a polynucleotide sequence which has at least 80% identity to thepolynucleotides as defined in (a) or (b) encoding a Caviidae allergen ora fragment thereof; (d) a polynucleotide sequence encoding a polypeptidewhich has at least 85% identity to the polypeptide as shown in SEQ IDNO:2 (Cav p 3), SEQ ID NO:6 (Cav p 2), SEQ ID NO:4 (Cav p 6) or SEQ IDNO:20 (Cavia porcellus allergen 1a) or a fragment thereof; (e) apolynucleotide sequence which hybridizes to the polynucleotide sequenceof any one of (a) to (d) and whereby the coding strand encodes aCaviidae allergen or a fragment thereof; (f) a polynucleotide sequenceencoding a polypeptide as encoded by the nucleotide sequence of any oneof (a) to (e) wherein at least one amino acid is deleted, substituted,inserted or added and whereby said polynucleotide encodes a Caviidaeallergen or a fragment thereof; and (g) a polynucleotide sequence beingdegenerate as a result of the generic code to the polynucleotidesequence as defined in any one of (a) to (f). The present invention alsoprovides for a method for producing the polypeptides (Cav p 2, Cav p 3,Cav p 6, Cavia porcellus allergen 1a) encoded by said polynucleotides.Moreover, the use of such allergens in a medical setting (e.g. in formof a pharmaceutical and/or diagnostic composition), an in vitro methodfor determining the allergenicity to Caviidae as well as an method foridentifying potential antangonists or agonists of the polypeptides arealso provided in the present invention.

One in four children and young adults in western countries produces IgEantibodies to common environmental antigens and is prone to developallergic diseases (Aberg (1995), Clin Exp Allergy 25, 815-9; Sears(1997), Lancet 350, 1015-1020; Beasly (1998), Lancet 351, 1225-1232).This trend to develop allergies is gaining worldwide relevance,affecting also “third-world-nations” hitherto supposed to be hardlyallergy-prone. Besides allergens of plant, invertebrate or mould origin,allergens of vertebrate origin are major triggers of type I allergicdisorders. From a respiratory point of view allergens linked to animaldander are of major importance. Up to 40% of atopic individuals have IgEto mammalian proteins (Ledford (1994), J Allergy Clin Immunol 94,327-334. Animals are important sources of inhalant allergens not only athome but also in public places and in working environments. One third toone half of households have mammalian pets. Cats and dogs, the mostpopular pets, are the most common cause of allergies to animals,followed by guinea-pigs and rabbits. But also hamsters, rats, and mice,or ferrets are kept as pets and can elicit allergies. Rodents are alsofrequent allergy triggers in laboratory workers (Bush (1998), J AllergyClin Immunol 102, 99-108). Occupational allergy to cow- andhorse-derived allergens affects mainly individuals, who are in directcontact with these animals: farmers, horse-owners, veterinarians.

Guinea pigs have a high sensitisation power provoking symptoms in 31% ofexposed laboratory workers in a Japanese study (Aoyama (1992) Br J IndMed 49, 41-47). In western countries most persons who become sensitizedto guinea-pigs become sensitized in their house-hold.

Conventional animal allergen extracts have been prepared from pelt,hair, dander, saliva, serum, urine. Although much work has been done onthe biochemical and immunochemical characterisation of these nativeallergen extracts, they are difficult to standardize in respect of thecontent of major and minor allergens and also the non-allergiccomponents. The quality and composition of the extracts are variable(Focke (2008), Clin Exp Allergy; 38(8):1400-8).

Commercial animal allergen extracts are generally prepared from hair ordander. They contain multiple components, allergenic as well asnon-allergenic molecules.

They are difficult to standardize in respect to the content of major andminor allergens.

Prick test solutions may be contaminated with products from othersources and give false positive results (Van der Veen (1996), J AllergyClin Immunol.; 98(6 Pt 1):1028-34). Today, standardization of allergenextracts for diagnosis and immunotherapy is based on biologicalstandardization like skin tests and competitive IgE binding assays. Inaddition, allergen manufacturers express potencies of their extracts incompany-specific units. This does not allow product comparison betweencompanies (Eichler (1988) Allergy; 43:458-63). For the diagnosis ofallergy to guinea pig, no purified native or recombinant molecules areavailable yet.

Allergic reaction to guinea pig has been recognized as a problem indomestic settings and work environments for many years. In the study ofFahlbusch (2002), Allergy 57, 417-422, one allergen of guinea pig, Cav p1, was characterized and the N-terminal amino-acid sequence wasdetermined.

Fahlbusch (2003), Allergy 58, 629-634 isolated and characterized afurther 17 kDa allergen from guinea pig hair which was termed Cav p 2.Furthermore, Fahlbusch eludicated allergenic as well as cross-reactiveproperties of the guinea pig allergens Cav p 1 and Cav p 2.

It was found in Fahlbusch (2003; loc. cit.) that Cav p 2 and Cav p 1contained several isoforms with pl values ranging from 3.6 to 5.3.Moreover, Fahlbusch (2003; loc. cit.) disclosed cross-reactive IgEepitopes on the allergens Cav p 2 and Cav p 1. However, guinea pigallergens are not characterized enough to allow standardization ofguinea pig extracts in terms of allergen content. There are no toolsavailable to quantify guinea pig allergens. Changing sources of primarymaterial as well as different methods for extract preparation mightresult in allergen preparations with variable allergen content. Howeverextracts with low amount of major allergens or lack of minor allergenswill loose sensitivity for diagnosis of guinea pig allergic patients.The isolation of pure molecules from guinea pig hair is an alternativeto using whole extracts. However this is laborious and time-consuming.

The technical problem underlying the present invention is the provisionof means and methods for diagnostic assessment and therapeuticintervention of allergenicity to Caviidae.

The technical problem is solved by provision of the embodimentscharacterized in the claims.

In a first aspect, the present invention relates to a nucleic acidmolecule encoding a Caviidae allergen comprising a polynucleotideselected from the group consisting of (a) a polynucleotide sequence asshown in SEQ ID NO:1 (Cav p 3), SEQ ID NO:5 (Cav p 2), SEQ ID NO:3 (Cavp 6) or SEQ ID NO: 19 (Cavia porcellus allergen 1a) or a fragmentthereof; (b) a polynucleotide sequence encoding a polypeptide as shownin SEQ ID NO:2 (Cav p 3), SEQ ID NO:6 (Cav p 2), SEQ ID NO:4 (Cav p 6)or SEQ ID NO: 20 (Cavia porcellus allergen 1a) or a fragment thereof;(c) a polynucleotide sequence which has at least 80% identity to thepolynucleotides as defined in (a) or (b) encoding a Caviidae allergen ora fragment thereof; (d) a polynucleotide sequence encoding a polypeptidewhich has at least 85% identity to the polypeptide as shown in SEQ IDNO:2 (Cav p 3), SEQ ID NO:6 (Cav p 2), SEQ ID NO:4 (Cav p 6) or SEQ IDNO:20 (Cavia porcellus allergen 1a) or a fragment thereof; (e) apolynucleotide sequence which hybridizes to the polynucleotide sequenceof any one of (a) to (d) and whereby the coding strand encodes aCaviidae allergen or a fragment thereof; (f) a polynucleotide sequenceencoding a polypeptide as encoded by the nucleotide sequence of any oneof (a) to (e) wherein at least one amino acid is deleted, substituted,inserted or added and whereby said polynucleotide encodes a Caviidaeallergen or a fragment thereof; and (g) a polynucleotide sequence beingdegenerate as a result of the generic code to the polynucleotidesequence as defined in any one of (a) to (f).

The present invention provides for novel antigens/allergens from guineapig Caviidae. Within the context the present invention, it has beensurprisingly found that purified native or recombinant guinea pigallergens/antigens as provided herein are particularly advantageous inthe diagnostic assessment and therapeutic intervention of allergenicity,in particular to Caviidae, as compared to corresponding conventionalextracts, like guinea pig extracts. Purified native guinea pigallergens/antigens as used herein and provided in this invention referpreferably to (a) polypeptide(s) or (a) fragment(s) thereof that is/areisolated from a cellular context (like the cellular context of a hostcell expression in a recombinant setting). In context of the appendedexamples it was in particular found that the submaxillary gland as wellas the harderian gland of guinea pig contain allergens according to thepresent invention, like Cav p 2, Cav p 3, Cav p6 and Cavia porcellusallergen 1a. The novel allergens/antigens are defined herein above andalso described herein in the sequences provided herein. It is to beunderstood that the present invention also provides for (a) fragment ofthe distinct sequences provided herein. Such fragments are in particularuseful in the preparation of e.g. antibody molecules directed againstthe novel and inventive guinea pig allergens/antigens, like in thepreparation of monoclonal or polyclonal antibodies. In this context inparticular fragments of at least 5 amino acids are useful. A furtheradvantageous use of fragments of the polypeptides of this invention inthis use in a medical setting, for example in immunization andsensitivisation approaches of (allergenic) patients. Obviously, alsofull length polypeptide of this invention can be used for diagnostic ormedical approaches provided herein.

In context of feature (c) herein above, it is envisaged thatpolynucleotides encoding for example a N-terminus of the correspondingCavia porcellus allergen 1a show an identity of at least 95%, 96%, 97%or 98% on DNA level. On the amino acid level an identity of at least 85%of said N-terminal fragment is within the scope of the presentinvention. Such (a) N-terminal fragment(s) comprise(s) at least 5 aminoacids and up to 15 amino acids or up to 18 amino acids with at least anidentity of 95%, 96%, 97% or 98%.

A particular preferred embodiment of the present invention is theprovision of a diagnostic tool in the determination of theimmunoglobulin level/titer (like the IgE-level/IgE-titer) in a subjectprone to suffer from an allergy or already suffering from an allergy. Inparticular, the determination of corresponding IgE levels/titers in asubject suffering from or prone to suffer from an allergy to Caviidae isenvisaged in context of the present invention. However, also other Igmay be determined, like IgG₁ or IgG₄. Corresponding working exampleshave been provided herein below and are also illustrated in the appendedexamples. In particular, the novel guinea pig antigens/allergensprovided herein and described in this invention constitute a majoradvantage in diagnostics settings over the “guinea pig extracts” thatare currently used in particular diagnostic settings. The presentinvention provides for highly specific diagnostic tools and specificcompounds to be used in medical settings, like immunization approachesand sensitivisation approaches of human patients. However, the presentinvention is not limited to medical intervention or diagnosis in humansbut also to other species like, for example, horses and other animals.As documented in the appended examples, a particular use of thepolypeptides of the present invention (or fragments thereof) is theiruse in diagnostic approaches which allow the discrimination between aspecific sensitization to a specific animal species (for examplesCaviidae) versus a cross-sensitization to other animals or animal parts.With the present invention it is now possible to produce isolated highlypurified proteins/allergens/antigens which can be obtained by forexample recombinant methods known in the art. Furthermore, such highlypurified or isolated proteins can be obtained routinely from animalextracts, like guinea pig extracts by the use of now available specificantibodies directed against the herein disclosed novel and inventiveguinea pig allergens/antigens. Protein purification methods fromextract, either recombinant extracts like recombinant extracts from hostcells or corresponding cell cultures or from guinea pig extracts arewell known in the art and can easily be applied by the person skilled inthe art. Within the means and methods provided herein is also adiagnostic method which allows the specific determination of IgEcross-reactivity, for example in biological samples from allergypatients. Such a biological sample may, for example be a blood sample,like a serum sample or a full blood sample. However, also skin samples,mucosal samples and the like may be used.

The gist of the present invention lies in the provision of novelCaviidae allergens. Moreover, it was surprisingly found that these novelallergens are particularly useful for diagnostic assessment andtherapeutic intervention of allergenicity to Caviidae, in particularguinea pigs. Furthermore, the use of combinations of said allergens isparticularly advantageous in the diagnostic assessment and therapeuticintervention of allergenicity to Caviidae, and accordingly envisaged inthe context of the present invention. The identification of Cav p 3, Cavp 6 and Cavia porcellus allergen 1a was not obvious as these proteinshave similar MW and pl and are difficult to separate by chromatography.In addition the amount of Cav p 3 and Cav p 6 protein found on hair islow. Proteins are more easily detected in the different gland extracts.The choice of the tissue was essential for isolation of Cav p 3, Cav p 6and Cavia porcellus allergen 1a. Yet, obtaining the allergens/antigensof the present invention is not a routine course of action which comeswithin the scope of the customary practice for the following reason:N-terminal amino acid sequence information allows the skilled person thedesign of degenerate primers which may be used in combination with anoligo(dT) primer to amplify the target mRNA by reverse transcription andpolymerase chain reaction. However, this approach failed for Caviaporcellus allergens as provided herein and in particular for the Caviaporcellus allergen 1a. Thus, a new strategy had to be developed. Thisstrategy is provided herein and is illustrated in the appended Examples,in particular Examples 8 and 9.

In one embodiment of the present invention a complementary strand of thenucleic acid molecule encoding a Caviidae allergen as defined herein isalso envisaged.

The meaning of the terms “nucleic acid molecule”, “polynucleotidesequence” and “polypeptide” is well known in the art, and the terms are,if not otherwise defined herein, used accordingly in the context of thepresent invention. For example, “polynucleotide sequence” as used hereinrefers to all forms of naturally occurring or recombinantly generatedtypes of nucleic acids and/or nucleotide sequences as well as tochemically synthesized nucleic acids/nucleotide sequences. This termalso encompasses nucleic acid analogs and nucleic acid derivatives suchas, e.g., locked DNA, PNA, oligonucleotide thiophosphates andsubstituted ribo-oligonucleotides. Furthermore, the term “polynucleotidesequence” also refers to any molecule that comprises nucleotides ornucleotide analogs.

Preferably, the term “polynucleotide sequence” refers to a nucleic acidmolecule, i.e. deoxyribonucleic acid (DNA) and/or ribonucleic acid(RNA). The “polynucleotide sequence” in the context of the presentinvention may be made by synthetic chemical methodology known to one ofordinary skill in the art, or by the use of recombinant technology, ormay be isolated from natural sources, or by a combination thereof. TheDNA and RNA may optionally comprise unnatural nucleotides and may besingle or double stranded. “Polynucleotide sequence” also refers tosense and anti-sense DNA and RNA, that is, a polynucleotide sequencewhich is complementary to a specific sequence of nucleotides in DNAand/or RNA. Furthermore, the term “polynucleotide sequence” may refer toDNA or RNA or hybrids thereof or any modification thereof that is knownin the state of the art (see, e.g., U.S. Pat. No. 5,525,711, U.S. Pat.No. 4,711,955, U.S. Pat. No. 5,792,608 or EP 302175 for examples ofmodifications). The polynucleotide sequence may be single- ordouble-stranded, linear or circular, natural or synthetic, and withoutany size limitation. For instance, the polynucleotide sequence may begenomic DNA, cDNA, mRNA, antisense RNA, ribozymal or a DNA encoding suchRNAs or chimeroplasts (Gamper, Nucleic Acids Research, 2000, 28,4332-4339). Said polynucleotide sequence may be in the form of a plasmidor of viral DNA or RNA. “Polynucleotide sequence” may also refer to (an)oligonucleotide(s), wherein any of the state of the art modificationssuch as phosphothioates or peptide nucleic acids (PNA) are included.

The term “allergen” is well known in the art. In context of the presentinvention, it refers in particular to a nonparasitic antigen capable ofstimulating a type-I hypersensitivity reaction in individuals withatopic syndrome. In individuals with atopic syndrome, non-parasiticantigens stimulate inappropriate IgE production, leading to type Ihypersensitivity. Sensitivities vary from one individual, preferablyhuman to another and it is possible to be allergic to an extraordinaryrange of substances. The type of allergens of the present inventioncomprises (an) allergen(s) derived/obtained from the class of mammalia,preferably rodentia, more preferably Caviidae, more preferably Caviinae,and even more preferably Cavia. Non-limiting examples of speciesbelonging to the genus Cavia are most preferably Cavia porcellus (guineapig(s)), Cavia anolaimae, Cavia guianae, Cavia aperea, Cavia fulgida,Cavia intermedia, Cavia magna, Cavia nana or Cavia tschudii. Caviaporcellus is, in context of the present invention, a preferred speciesof the family Caviidae. In a preferred embodiment, the allergen is apolypeptide as described and defined herein below, in particular apolypeptide as shown in SEQ ID NO: 2 (Cav p 3), SEQ ID NO: 6 (Cav p 2),SEQ ID NO: 4 (Cav p 6) or SEQ ID NO: 20 (Cavia porcellus allergen 1a).Accordingly, a polypeptide is disclosed herein, wherein said polypeptideis selected from a group consisting of (a) a polypeptide comprising anamino acid sequence encoded by a nucleic acid molecule having thenucleic acid sequence as depicted in SEQ ID NO: 1 (Cav p 3), SEQ ID NO:5 (Cav p 2), SEQ ID NO: 3 (Cav p 6) or SEQ ID NO: 19 (Cavia porcellusallergen 1a); (b) a polypeptide having an amino acid sequence asdepicted in SEQ ID NO: 2 (Cav p 3), SEQ ID NO: 6 (Cav p 2), SEQ ID NO: 4(Cav p 6) or SEQ ID NO: 20 (Cavia porcellus allergen 1a); (c) apolypeptide comprising an amino acid encoded by a nucleic acid moleculeencoding a peptide having an amino acid sequence as depicted in SEQ IDNO: 2 (Cav p 3), SEQ ID NO: 6 (Cav p 2), SEQ ID NO: 4 (Cav p 6) or SEQID NO: 20 (Cavia porcellus allergen 1a).; (d) a polypeptide comprisingan amino acid encoded by a nucleic acid molecule hybridizing understringent conditions to the complementary strand of nucleic acidmolecules as defined in (a) or (c) and encoding a Caviidae allergen or afragment thereof; (e) a polypeptide having at least 85% identity to thepolypeptide of any one of (a) to (d), and encoding a Caviidae allergenor a fragment thereof; and (f) a polypeptide comprising an amino acidencoded by a nucleic acid molecule having a polynucleotide sequence of anucleic acid molecule as defined in (a), (c), (d) and (e).

The term “fragment thereof” as used herein in context of polypeptides,refers to a functional fragment which has essentially the same(biological) activity as the polypeptides defined herein (e.g. as shownin Seq ID NOs 2, 4, 6 or 20, respectively) which may be) encoded by thepolynucleotides of the present invention (e.g. Seq ID NOs 1, 3, 5 or 19,respectively). With respect to a nucleotide sequence of Caviidaeallergen, the term “fragment” as used herein means a nucleotide sequencebeing at least 7, at least 10, at least 15, at least 20, at least 30, atleast 50, at least 100, at least 150, at least 200 or at least 250nucleotides in length. As discussed herein above, an inventive andillustrative N-terminal fragment of the polypeptide provided in SEQ IDNO: 20 is a fragment that shows at least an identity of 95% to the first15 amino acids as provided in SEQ ID NO: 20. Further sequence identityalso in context of the other polypeptides of this invention, wereprovided herein above. With respect to an amino acid sequence ofCaviidae allergen, the term “fragment” as used herein exemplary means anamino acid sequence being of at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20 or at least 25 amino acid residues in length. Onefragment according to this invention comprises at least 5 amino acidsand ranges up to the first 15 amino acids or up to at least 18 aminoacids of the N-terminal-region of Cavia porcellus allergen 1a as alsodisclosed in FIG. 20.

The person skilled in the art is in the position to deduce the codingsequences of the novel Caviidae allergens provided herein. The codingregions as well as the proteinous structures/amino acids are providedherein and, for example, for Cavia porcellus allergen 1a in FIG. 20, thestart codon is at nt 55-57, the stop codon is at nt 553-555; for Cav p 2in FIG. 4 start codon is at nt 53-55 and stop codon is at nt 563-565;for Cav p 3 in FIG. 8 start codon is at nt 59-61 and stop codon is at nt569-571; for Cav p 6 in FIG. 10 start codon is at nt 92-94 and stopcodon is at nt 635-637.

The term “activity” as used herein refers in particular to thecapability of (a) polypeptide(s) or (a) fragment(s) thereof to elicit anallergic response. In other words, the polypeptide(s) or fragment(s)thereof provided in the present invention are (an) allergen(s) asdescribed herein above, preferably Caviidae allergen(s), more preferably(a) Cavia allergen(s) and most preferably (a) Cavia porcellus (guineapig) allergen. It is to be understood that (an) allergen(s) (e.g. (a)guinea pig allergen(s)) is capable of eliciting an allergic response tosaid allergen not only in a human, but also in other animals, inparticular in animals in which the allergen(s) are not/cannot bederived/obtained from (for example, in non-rodents, such as dogs, catsand the like). The definitions and explanations given herein below incontext of diagnosis and treatment of patients suffering from (orsuspected of suffering from) an allergy to (an) Caviidae allergen(s)also apply, mutatis mutandis, to the animal(s) described above.Furthermore, also envisaged in the present invention is an animal modelwhich can be used for experimental immunotherapy.

A person skilled in the art will be aware that the (biological) activityas described herein often correlates with the expression level (e.g.protein/mRNA). If not mentioned otherwise, the term “expression” usedherein refers to the expression of a nucleic acid molecule encoding apolypeptide/protein (or a fragment thereof) of the invention, whereas“activity” refers to activity of said polypeptide/protein.Methods/assays for determining the activity of polypeptides (e.g. thepolypeptides shown in Seq ID Nos 2, 4, 6 or 20) are well known in theart. Exemplary methods for assessing the activity of polypeptides of thepresention invention are shown in Example 6 (e.g. testing for IgEbinding in immunoblots, ELISA for IgE reactivity). In context ofpolynucleotide sequences the term “fragment thereof” refers inparticular to (a) fragment(s) of nucleic acid molecules, wherein saidnucleic acid molecules preferably encode (a) Caviidae allergen(s) (or afragment thereof) as defined above. A “fragment of a polynucleotide”may, for example, encode a polypeptide of the present invention (e.g. apolypeptide as shown in SEQ ID NOs 2, 4, 6 or 20) having at least oneamino acid deletion and, optionally, at least one amino acidsubstitution, whereby said polypeptide preferably is a Caviidaeallergen. Such a shortened polypeptide may be considered as a functionalfragment of a polypeptide of the present invention (e.g. as shown in SEQID NOs 2, 4, 6 or 20).

The term “hybridizes” used herein may refer to hybridization underconventional hybridization conditions, preferably under stringentconditions, as for instance described in Sambrook and Russell (2001),Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor,N.Y., USA. If not further specified, the conditions are preferablynon-stringent. Said hybridization conditions may be establishedaccording to conventional protocols described, e.g., in Sambrook (2001)loc. cit. The setting of conditions is well within the skill of theartisan and can be determined according to protocols described in theart. Thus, the detection of only specifically hybridizing sequences willusually require stringent hybridization and washing conditions. As anon-limiting example, highly stringent hybridization may occur under thefollowing conditions:

-   Hybridization buffer: 2×SSC; 10×Denhardt solution (Fikoll    400+PEG+BSA; ratio 1:1:1); 0.1% SDS; 5 mM EDTA; 50 mM Na₂HPO₄;-    250 μg/ml of herring sperm DNA; 50 μg/ml of tRNA; or-    0.25 M of sodium phosphate buffer, pH 7.2;-    1 mM EDTA-    7% SDS-   Hybridization temperature T=60° C.-   Washing buffer: 2×SSC; 0.1% SDS-   Washing temperature T=60° C.

Low stringent hybridization conditions for the detection of homologousor not exactly complementary sequences may, for example, be set at6×SSC, 1% SDS at 65° C. As is well known, the length of the probe andthe composition of the nucleic acid to be determined constitute furtherparameters of the hybridization conditions.

Polynucleotide sequences which are capable of to hybridizing with thepolynucleotide sequences provided herein can for instance be isolatedfrom genomic libraries or cDNA libraries of animals. Preferably, suchpolynucleotides are from animal origin, particularly preferred from ananimal belonging to the class of mammalia, preferably rodentia, morepreferably Caviidae, more preferably Caviinae, more preferably Cavia,most preferably Cavia porcellus (guinea pig(s)), Cavia anolaimae, Caviaguianae, Cavia aperea, Cavia fulgida, Cavia intermedia, Cavia magna,Cavia nana or Cavia tschudii. Alternatively, such nucleotide sequencescan be prepared by genetic engineering or chemical synthesis.

Such polynucleotide sequences being capable of hybridizing may beidentified and isolated by using the polynucleotide sequences describedherein or parts or reverse complements thereof, for instance byhybridization according to standard methods (see for instance Sambrookand Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press,Cold Spring Harbor, N.Y., USA). Nucleotide sequences comprising the sameor substantially the same nucleotide sequences as indicated in thelisted SEQ ID NOs, or parts/fragments thereof, can, for instance, beused as hybridization probes. The fragments used as hybridization probescan also be synthetic fragments which are prepared by usual synthesistechniques, the sequence of which is substantially identical with thatof a nucleotide sequence according to the invention.

The meaning of the term “identity” and “homology”, respectively,particularly with respect to two nucleotide sequences or amino acidsequences/polypeptides to be compared, is known in the art and usedherein accordingly. For example, this term is used herein in the contextof a nucleotide sequence or amino acid sequence/polypeptide which has ahomology, that is to say a sequence identity, of at least 50%, 55%, 60%,preferably of at least 70%, 75% more preferably of at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94, 95%, 96%, 97%, 98%, and evenmost preferably of at least 99% to another, preferably entire,nucleotide sequence or amino acid sequence, respectively. In otherwords, a polypeptide (being a Caviidae allergen or fragment thereof) hasat least 50%, 55% 60% preferably at least 70%, 75% more preferably atleast 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94, 95%, 96%,97%, 98%, and most preferably at least 99% identity/homology to thepolypeptide shown in Seq ID No 2, 4, 6 and 20, respectively.

If the two nucleotide sequences to be compared by sequence comparisonsdiffer in identity refers to the shorter sequence and that part of thelonger sequence that matches the shorter sequence. In other words, whenthe sequences which are compared do not have the same length, the degreeof identity preferably either refers to the percentage of nucleotideresidues in the shorter sequence which are identical to nucleotideresidues in the longer sequence or to the percentage of nucleotides inthe longer sequence which are identical to nucleotide sequence in theshorter sequence. In this context, the skilled person is readily in theposition to determine that part of a longer sequence that “matches” theshorter sequence. The search for sequence identity or homology on DNAand protein level can be done according to the present invention byusing the publically available program Blast. Further, pairwise sequencecomparisons may be done using Gap on the Heidelberg Unix SequenceAnalysis Resources (HUSAR) server using the alignment method ofNeedleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970).

In general, the person skilled in the art knows how nucleic acidmolecules can be obtained, for instance, from natural sources (e.g.animals belonging to the family “Caviidae”) or may also be producedsynthetically or by recombinant techniques, such as PCR. These nucleicacid molecules and include modified or derivatized, nucleic acidmolecules as can be obtained by applying techniques described in thepertinent literature. Primers as provided herein are useful indiagnostic as well as scientific (research) settings. Accordingly, thepresent invention also provides defined primer as shown in SEQ ID NOS: 7to 11 for the amplification of the Cav p 2 gene, SEQ ID NOS: 12 to 16for the amplification of the Cav p 2 gene, SEQ ID NOS: 17 to 18 for theamplification of the Cav p 6 gene and SEQ ID NOS: 21 to 42 for theamplification of the Cavia porcellus allergen 1a gene or a fragmentthereof as shown in SEQ ID NO:1 (Cav p 3), SEQ ID NO:5 (Cav p 2), SEQ IDNO:3 (Cav p 6) or SEQ ID NO: 19 (Cavia porcellus allergen 1a). Theseprimers can be also used as primer pairs within the present invention.

The primers being represented by SEQ ID NOS: 21 to 32 (Cavp-MQ1 toCavp-MQ 12) and SEQ ID NOS: 41 to 42 (Cavp-11 for (3′RACE) andCavp-b_rev for (5′RACE)) bind to both isoforms of the Caviidae allergensCavia porcellus allergen 1a gene and Cav p 1. However, the primers beingrepresented by SEQ ID NOS: 33 to 40 (Cavp-MQ1rev to Cavp-MQ8rev) bindspecifically to the Cavia porcellus allergen 1a gene. These primers canbe further used in a kit according to the invention. The primer usedwithin the present invention as shown in SEQ ID NOS: 7 to 11, 12 to 16,17 to 18 and 21 to 42 may also comprise a shorter sequence starting fromAAR to TGG with 18 nucleotides. However, the skilled person is in aposition to modify and amend this primers, primer sequences and primerpairs, for example, by elongation and/or shortening of the sequences ordistinct nucleotide exchanges. Such an exchange needs to lead to acomplementary strand sequence that is still capable to specificallyhybridize under conditions disclosed herein (for example stringenthybridization conditions) to its corresponding sequence on thecorresponding complementary strand.

Identity, moreover, means that there is a functional and/or structuralequivalence between the corresponding nucleotide sequence orpolypeptides, respectively (e.g. polypeptides encoded thereby).Nucleotide/amino acid sequences which have at least 50%, 55%, 60%,preferably of at least 70%, 75% more preferably of at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94, 95%, 96%, 97%, 98%, and evenmost preferably of at least 99% identity to the herein-describedparticular nucleotide/amino acid sequences may representderivatives/variants of these sequences which, preferably, have the samebiological function. They may be either naturally occurring variations,for instance sequences from other ecotypes, varieties, species, etc., ormutations, and said mutations may have formed naturally or may have beenproduced by deliberate mutagenesis. Furthermore, the variations may besynthetically produced sequences. The allelic variants may be naturallyoccurring variants or synthetically produced variants or variantsproduced by recombinant DNA techniques. Deviations from theabove-described polynucleotides may have been produced, e.g., bydeletion, substitution, addition, insertion and/or recombination. Theterm “addition” refers to adding at least one nucleic acid residue/aminoacid to the end of the given sequence, whereas “insertion” refers toinserting at least one nucleic acid residue/amino acid within a givensequence.

The variant polypeptides and, in particular, the polypeptides encoded bythe different variants of the nucleotide sequences of the inventionpreferably exhibit certain characteristics they have in common. Theseinclude, for instance, biological activity, molecular weight,immunological reactivity, conformation, etc., and physical properties,such as for instance the migration behavior in gel electrophoreses,chromatographic behavior, sedimentation coefficients, solubility,spectroscopic properties, stability, pH optimum, temperature optimumetc.

The present invention provides for antigens/allergens from Caviideawhich are particularly useful in their isolated and purified form fordiagnostic purposes disclosed herein as well as in pharmaceuticalcompositions. The term “isolated” as used herein with reference to anucleic acid molecule refers to a nucleic acid molecule that isseparated from at least one other nucleic acid molecule with which it isordinarily associated, for example, in its natural environment. Anisolated nucleic acid molecule further includes a nucleic acid moleculecontained in cells that ordinarily express the nucleic acid molecule,but the nucleic acid molecule is present extrachromosomally or at achromosomal location that is different from its natural chromosomallocation. Isolated nucleic acid molecules also refer to molecules thatare substantially free of material from the environment of production.For example, isolated nucleic molecules refer to molecules free ofcellular material and culture medium/products when produced byrecombinant methods, or chemical precursors or other reaction chemicalswhen synthesized.

The term “isolated” as used herein with reference to a protein refers toa protein that is separated from at least one other protein with whichit is ordinarily associated, for example, in its natural environment.Isolated proteins also refer to molecules that are substantially free ofmaterial from the environment of production. For example, isolatedproteins are substantially free of cellular material and culturemedium/products when produced by recombinant methods, or chemicalprecursors or other reaction chemicals when synthesized. “Purified” asused herein references that a molecule of the present invention(including the herein disclosed polypeptides or fragments thereof) ispresent in a sample at a concentration of at least 90% by weight,preferably at least 95% by weight, and more preferably at least 98% byweight of the sample in which it is contained.

Accordingly, and in a further aspect, the present invention relates to avector comprising the nucleic acid molecules described herein and arecombinant host cell comprising the nucleic acid molecules and/or thevector.

The term “vector” as used herein particularly refers to plasmids,cosmids, viruses, bacteriophages and other vectors commonly used ingenetic engineering. In a preferred embodiment, the vectors of theinvention are suitable for the transformation of cells, like fungalcells, cells of microorganisms such as yeast or bacterial cells oranimal cells. The vectors as well as the host cells of the presentinvention are particularly useful in the recombinant expression of thepolypeptides (Caviidae allergens/antigens and fragments thereof) of thepresent invention.

In a further aspect, the recombinant host cell of the present inventionis capable of expressing or expresses the polypeptide encoded by thepolynucleotide sequence of this invention. In a specific embodiment, the“polypeptide” comprised in the host cell may be a heterologous withrespect to the origin of the host cell. An overview of examples ofdifferent expression systems to be used for generating the host cell ofthe present invention, for example the above-described particular one,is for instance contained in Glorioso et al. (1999), Expression ofRecombinant Genes in Eukaryotic Systems, Academic Press Inc.,Burlington, USA, Paulina Balbas und Argelia Lorence (2004), RecombinantGene Expression: Reviews and Protocols, Second Edition: Reviews andProtocols (Methods in Molecular Biology), Humana Press, USA.

The transformation or genetically engineering of the host cell with anucleotide sequence or the vector according to the invention can becarried out by standard methods, as for instance described in Sambrookand Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press,Cold Spring Harbor, N.Y., USA. Moreover, the host cell of the presentinvention is cultured in nutrient media meeting the requirements of theparticular host cell used, in particular in respect of the pH value,temperature, salt concentration, aeration, antibiotics, vitamins, traceelements etc.

The term “recombinant host cell”, as used herein, relates to a hostcell, genetically engineered with the nucleotide sequence of the presentinvention or comprising the vector or the polypeptide or a fragmentthereof of the present invention.

Generally, the host cell of the present invention may be a prokaryoticor eukaryotic cell comprising the nucleotide sequence, the vector and/orthe polypeptide of the invention or a cell derived from such a cell andcontaining the nucleotide sequence, the vector and/or the polypeptide ofthe invention. In a preferred embodiment, the host cell comprises, forexample due to genetic engineering, the nucleotide sequence or thevector of the invention in such a way that it contains the nucleotidesequences of the present invention integrated into the genome.Non-limiting examples of such a host cell of the invention (but also thehost cell of the invention in general) may be a bacterial, yeast,fungus, plant, animal or human cell.

In accordance with the above, the invention relates in a furtherembodiment to a method for producing a polypeptide provided herein,comprising culturing the recombinant host cell under such conditionsthat the polypeptide is expressed, and recovering the polypeptide.

The term “such conditions”, as used herein, refers to culture conditionsof recombinant host cells in order to express and recover polypeptides,preferably heterologous polypeptides. These conditions are well known toa person skilled in the art, as for instance described in Sambrook andRussell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, ColdSpring Harbor, N.Y., USA.

In general, the polypeptide of this invention may, in addition to theCaviidae allergen of the invention and as defined herein, comprise (a)further polypeptide(s), i.e. (a) polypeptide(s) being heterologous withrespect to the polypeptides of the present invention. One skilled in theart will be aware that the heterologous polypeptide can be expressed byprokaryotic (e.g. bacteria) or eukaryotic cell (e.g. 293 cells or CHOcells). The present invention also relates to a fusion protein (and anucleic acid molecule encoding the fusion protein). For example, thefurther/heterologous polypeptide(s) may particularly be suitable for apotentiated or increased production for the polypeptide of the presentinvention (for example, in a cell). The further/heterologouspolypeptide(s) may, for example, comprise a protein fragment or peptide,an entire functional moiety, or an entire protein sequence which can bedesigned to be used in purifying the fusion protein, for example eitherwith antibodies or with affinity purification specific for thefurther/heterologous polypeptide. Likewise, physical properties of theadditional polypeptide, protein fragment, peptide (and the like) can beexploited to allow selective purification of the heterologouspolypeptide and, hence, also the polypeptide of the present invention.

As a fusion protein, the polypeptides of the present invention as shownin Seq ID No 2, 4, 6 and 20, respectively, may also include a reporteror reporter construct being expressible in a cell, a tissue, a cellculture, tissue culture, animals or plants. Thus, the polypetide of thepresent invention can be easily identified and measured by the skilledperson, in order to determine whether the polypeptide has been expressedin the cell, animals or plants

Commonly used reporter genes that induce visually identifiablecharacteristics usually involve fluorescent and luminescent proteins;examples include the gene that encodes jellyfish green fluorescentprotein (GFP), which causes cells that express it to glow green underblue light, the enzyme luciferase, which catalyzes a reaction withluciferin to produce light, and the red fluorescent protein from thegene dsRed. Another common reporter in bacteria is the lacZ gene, whichencodes the protein β-galactosidase. This enzyme causes bacteriaexpressing the gene to appear blue when grown on a medium that containsthe substrate analog X-gal (an inducer molecule such as Isopropylβ-D-1-thiogalactopyranoside (IPTG) is also needed under the nativepromoter).

One of skill in the art will recognize that the particularpeptide/protein fragment etc. is chosen with the purification scheme inmind. As a non-limiting example, His tags, GST, and maltose-bindingprotein represent peptides that have readily available affinity columnsto which they can be bound and eluted. Thus, where the peptide is anN-terminal His tag such as hexahistidine (His.sub.6 tag), theheterologous protein can be purified using a matrix comprising ametal-chelating resin, for example, nickel nitrilotriacetic acid(Ni-NTA), nickel iminodiacetic acid (Ni-IDA), and cobalt-containingresin (Co-resin). See, for example, Steinert et al. (1997) QIAGEN News4:11-15. Where the peptide is GST, the heterologous protein can bepurified using a matrix comprising glutathione-agarose beads (Sigma orPharmacia Biotech); where the protein fragment is a maltose-bindingprotein (MBP), the heterologous protein can be purified using a matrixcomprising an agarose resin derivatized with amylose.

In a further embodiment, the present invention refers to a polypeptideor fragment thereof having the amino acid sequence encoded by one of thenucleic acid molecules which encodes a Caviidae allergen. Furthermore,the polypeptide is obtainable by the above-mentioned method.Polypeptides of the present invention have been described in detailherein above.

Disclosed herein are molecules which bind to the nucleic acid moleculesand/or polypeptides of the present invention and which can, accordinglyused in the herein described uses and methods. Binding moleculesaccording to the invention can, inter alia, be used for detecting thepresence, absence or amount of the nucleic acid molecules/polypeptidesof the invention in a sample, in particular in the framework of methodsand uses described herein. The binding molecules may furthermore be usedfor isolating the nucleic acid molecules/polypeptides from a biologicalsource material. Moreover, the binding molecules to be used/to beidentified in accordance with the invention may be used toinhibit/antagonize Cav p 2, Cav p 3, Cav p 6 and/or Cavia porcellusallergen 1a or fragments thereof, which may, for example, result in areduction of the amount or inhibition of the activity of themolecules/polypeptides as defined herein.

Non-limiting examples of suitable binding molecules may be selected fromaptamers (Klussmann (2006), The Aptamer Handbook: FunctionalOligonucleotides and their applications, Wiley-VCH, USA), RNAi, shRNA(McIntyre and Fanning (2006), BMC Biotechnology 6:1), ribozymes,antisense nucleotide sequences (like antisense DNAs or antisense RNAs),siRNA (Hannon (2003), Rnai: A Guide to Gene Silencing, Cold SpringHarbor Laboratory Press, USA), PNAs, antibodies (Howard and Bethell(2000) Basic Methods in Antibody Production and Characterization, Crc.Pr. Inc), (Hansson, Immunotechnology 4 (1999), 237-252; Henning, HumGene Ther. 13 (2000), 1427-1439), affibodies, lectins, trinectins(Phylos Inc., Lexington, Mass., USA; Xu, Chem. Biol. 9 (2002), 933),anticalins (EPB1 1 017 814) and the like.

In a particular embodiment, the present invention relates to an antibodyand the use thereof that specifically binds to the polypeptide orfragments thereof as shown in Seq ID No 2, 4, 6 and 20, respectively,and as further described and defined herein, in particular a Caviidaeallergen. Moreover, said antibody can be used for the purification ofsaid polypeptide. The term “antibody” is well known in the art.

In context of the present invention, the term “antibody” as used hereinrelates in particular to full immunoglobulin molecules as well as toparts of such immunoglobulin molecules substantially retaining bindingspecificity. Furthermore, the term relates to modified and/or alteredantibody molecules, like chimeric and humanized antibodies, CDR-graftedantibodies, recombinantly or synthetically generated/synthesizedantibodies and to intact antibodies as well as to antibody fragmentsthereof, like, separated light and heavy chains, Fab, Fab/c, Fv, Fab′,F(ab′)₂. The term “antibody” also comprises bifunctional antibodies,trifunctional antibodies and antibody constructs, like single chain Fvs(scFv) or antibody-fusion proteins. Further “antibody” constructs areknown in the art and comprised in the present invention.

Techniques for the production of antibodies are well known in the artand described, e.g. in Howard and Bethell (2000) Basic Methods inAntibody Production and Characterization, Crc. Pr. Inc. Antibodiesdirected against a polypeptide according to the present invention can beobtained, e.g., by direct injection of the polypeptide (or a fragmentthereof) into an animal or by administering the polypeptide (or afragment thereof) to an animal. The antibody so obtained will then bindpolypeptide (or a fragment thereof) itself. In this manner, even afragment of the polypeptide can be used to generate antibodies bindingthe whole polypeptide, as long as said binding is “specific” as definedabove.

These polypeptides are particularly useful in the preparation ofspecific antibodies and are provided herein for illustrative purposes.

Also in the appended examples (e.g. Example 2) the generation ofspecific antibodies against a polypeptide of the present invention areshown. In case of Cavia porcellus allergen 1a, potential epitopes forproduction of specific antibodies to Cavia porcellus allergen 1a are,for example, shown in SEQ 20 at positions 78-86, 106-110 and/or 139-153.

With the normal skill of the person skilled in the art and by routinemethods, the person skilled in the art can easily deduce from thesequences provided herein relevant epitopes (also functional fragments)of the polypeptides of the present invention which are useful in thegeneration of antibodies like polyclonal and monoclonal antibodies.However, the person skilled in the art is readily in a position to alsoprovide for engineered antibodies like CDR-grafted antibodies or alsohumanized and fully human antibodies and the like.

Particularly preferred in the context of the present invention aremonoclonal antibodies. For the preparation of monoclonal antibodies, anytechnique which provides antibodies produced by continuous cell linecultures can be used. Examples for such techniques include the hybridomatechnique, the trioma technique, the human B-cell hybridoma techniqueand the EBV-hybridoma technique to produce human monoclonal antibodies(Shepherd and Dean (2000), Monoclonal Antibodies: A Practical Approach,Oxford University Press, Coding and Coding (1996), MonoclonalAntibodies: Principles and Practice—Production and Application ofMonoclonal Antibodies in Cell Biology, Biochemistry and Immunology,Academic Pr Inc, USA).

The antibody derivatives can also be produced by peptidomimetics.Further, techniques described for the production of single chainantibodies (see, inter alia, U.S. Pat. No. 4,946,778) can be adapted toproduce single chain antibodies specifically recognizing the polypeptideof the invention. Also, transgenic animals may be used to expresshumanized antibodies to the polypeptide of the invention.

As shown in the appended examples, the present invention also envisagesthe production of specific antibody against native polypeptides andrecombinant polypeptides according to the invention. This production isbased, for example, on the immunization of animals, like mice. However,also other animals for the production of antibody/antisera is envisagedwithin the present invention. For example, monoclonal and polyclonalantibodies can be produced by rabbit, mice, goats, donkeys and the like.The polynucleotide according to the invention as shown in SEQ ID NO:1(Cav p 3), SEQ ID NO:5 (Cav p 2), SEQ ID NO:3 (Cav p 6) or SEQ ID NO: 19(Cavia porcellus allergen 1a) can be subcloned into an appropriatedvector, wherein the recombinant polypeptide is to be expressed in anorganism being able for an expression, for example in bacteria. Thus,the expressed recombinant protein can be intra-peritoneally injectedinto a mice and the resulting specific antibody can be, for example,obtained from the mice serum being provided by intra-cardiac bloodpuncture. The amount of obtained specific antibody can be quantifiedusing an ELISA, which is also described herein below. Further methodsfor the production of antibodies are well known in the art, see, e.g.Harlow and Lane, “Antibodies, A Laboratory Manual”, CSH Press, ColdSpring Harbor, 1988.

The term “specifically binds”, as used herein, refers to a bindingreaction that is determinative of the presence of the Caviidae allergenand antibody in the presence of a heterogeneous population of proteinsand other biologics. Also, the term “specifically binds” relates to, forexample, for the distinction between the Cavp 1 (N-Terminus as providedby Fahlbusch loc. cit.) and the novel Cavia porcellus allergen 1a geneas provided herein.

Thus, under designated assay conditions, the specified antibodies andallergens bind to one another and do not bind in a significant amount toother components present in a sample. Specific binding to a targetanalyte under such conditions may require a binding moiety that isselected for its specificity for a particular target analyte. A varietyof immunoassay formats may be used to select antibodies specificallyreactive with a particular antigen. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with an analyte. See Shepherd and Dean(2000), Monoclonal Antibodies: A Practical Approach, Oxford UniversityPress and/or Howard and Bethell (2000) Basic Methods in AntibodyProduction and Characterization, Crc. Pr. Inc. for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity. Typically a specific or selective reactionwill be at least twice background signal to noise and more typicallymore than 10 to 100 times greater than background. The person skilled inthe art is in a position to provide for and generate specific bindingmolecules directed against the novel polypeptides. For specificbinding-assays it can be readily employed to avoid undesiredcross-reactivity, for example polyclonal antibodies can easily bepurified and selected by known methods (see Shepherd and Dean, loc.cit.).

The term “purification”, as used herein, refers to a series of processesintended to isolate a single type of protein from a complex mixture.Protein purification is vital for the characterisation of the function,structure and interactions of the protein of interest. The startingmaterial, as a non-limiting example, can be a biological tissue or amicrobial culture. The various steps in the purification process mayfree the protein from a matrix that confines it, separate the proteinand non-protein parts of the mixture, and finally separate the desiredprotein from all other proteins. Separation steps exploit differences inprotein size, physico-chemical properties and binding affinity.Exemplary purification methods are also shown in appended Example 1.

In accordance with the above and in relation with the embodiments ofthis invention, the present invention relates also to a pharmaceuticalcomposition comprising the polynucleotide as shown in SEQ ID NO:1 (Cav p3), SEQ ID NO:5 (Cav p 2), SEQ ID NO:3 (Cav p 6) or SEQ ID NO: 19 (Caviaporcellus allergen 1a), the vector, the polypeptide as shown in Seq IDNo 2, 4, 6 and 20, respectively, or the antibody. The pharmaceuticalcomposition of the present invention may also comprise (functional)fragments of the polypeptides provided herein. Such (functional)fragments may, inter alia, be used in the modification of the immunesystem of a patient, like an allergenic patient. Moreover, the use ofthe pharmaceutical composition in active or passive immunisation, likehyposensiblisation or in medical intervention of an allergic reaction isalso envisaged in the context of the present invention. Activeimmunization means in this context to the introduction of a foreignmolecule into the body, which causes the body itself to generateimmunity against the target. It clear for the skilled person that thisimmunity comes from the T cells and the B cells with their antibodies.Passive immunization relates to an immunization where pre-synthesizedelements of the immune system are transferred to a person so that thebody does not need to produce these elements itself. Artificial passiveimmunization is normally administered by injection and is used if therehas been a recent outbreak of a particular disease.

The pharmaceutical composition will be formulated and dosed in a fashionconsistent with good medical practice, taking into account the clinicalcondition of the individual patient, the site of delivery of thepharmaceutical composition, the method of administration, the schedulingof administration, and other factors known to practitioners. The“effective amount” of the pharmaceutical composition for purposes hereinis thus determined by such considerations.

The skilled person knows that the effective amount of pharmaceuticalcomposition administered to an individual will, inter alia, depend onthe nature of the compound. For example, if said compound is a(poly)peptide or protein the total pharmaceutically effective amount ofpharmaceutical composition administered parenterally per dose will be inthe range of about 1 pg protein/kg/day to 10 mg protein/kg/day ofpatient body weight, although, as noted above, this will be subject totherapeutic discretion. More preferably, this dose is at least 0.01 mgprotein/kg/day, and most preferably for humans between about 0.01 and 1mg protein/kg/day. If given continuously, the pharmaceutical compositionis typically administered at a dose rate of about 1 μg/kg/hour to about50 μg/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed. The length of treatment needed toobserve changes and the interval following treatment for responses tooccur appears to vary depending on the desired effect. The particularamounts may be determined by conventional tests which are well known tothe person skilled in the art.

Pharmaceutical compositions of the invention may be administered orally,parenterally, intracisternally, intraperitoneally, topically (as bypowders, ointments, drops or transdermal patch), bucally, or as an oralor nasal spray.

Pharmaceutical compositions of the invention preferably comprise apharmaceutically acceptable carrier. By “pharmaceutically acceptablecarrier” is meant a non-toxic solid, semisolid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.The term “parenteral” as used herein refers to modes of administrationwhich include intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intraarticular injection and infusion.

The pharmaceutical composition is also suitably administered bysustained release systems. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or mirocapsules. Sustained-releasematrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. etal., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate)(R. Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and R.Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langeret al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustainedrelease pharmaceutical composition also include liposomally entrappedcompound. Liposomes containing the pharmaceutical composition areprepared by methods known per se: DE 3,218,121; Epstein et al., Proc.Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl.Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046;EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos.4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes areof the small (about 200-800 Angstroms) unilamellar type in which thelipid content is greater than about 30 mol. percent cholesterol, theselected proportion being adjusted for the optimal therapy.

For parenteral administration, the pharmaceutical composition isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation.

Generally, the formulations are prepared by contacting the components ofthe pharmaceutical composition uniformly and intimately with liquidcarriers or finely divided solid carriers or both. Then, if necessary,the product is shaped into the desired formulation. Preferably thecarrier is a parenteral carrier, more preferably a solution that isisotonic with the blood of the recipient. Examples of such carriervehicles include water, saline, Ringer's solution, and dextrosesolution. Non aqueous vehicles such as fixed oils and ethyl oleate arealso useful herein, as well as liposomes. The carrier suitably containsminor amounts of additives such as substances that enhance isotonicityand chemical stability. Such materials are non-toxic to recipients atthe dosages and concentrations employed, and include buffers such asphosphate, citrate, succinate, acetic acid, and other organic acids ortheir salts; antioxidants such as ascorbic acid; low molecular weight(less than about ten residues) (poly)peptides, e.g., polyarginine ortripeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids, such as glycine, glutamic acid, aspartic acid, or arginine;monosaccharides, disaccharides, and other carbohydrates includingcellulose or its derivatives, glucose, manose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;counterions such as sodium; and/or nonionic surfactants such aspolysorbates, poloxamers, or PEG.

The components of the pharmaceutical composition to be used fortherapeutic administration must be sterile. Sterility is readilyaccomplished by filtration through sterile filtration membranes (e.g.,0.2 micron membranes). Therapeutic components of the pharmaceuticalcomposition generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The components of the pharmaceutical composition ordinarily will bestored in unit or multi-dose containers, for example, sealed ampoules orvials, as an aqueous solution or as a lyophilized formulation forreconstitution. As an example of a lyophilized formulation, 10-ml vialsare filled with 5 ml of sterile-filtered 1% (w/v) aqueous solution, andthe resulting mixture is lyophilized. The infusion solution is preparedby reconstituting the lyophilized compound(s) using bacteriostaticWater-for-Injection.

The term “medical intervention” as herein described, refers to anyexamination, treatment, or other act having preventive, diagnostictherapeutic or rehabilitative aims and which is carried out by aphysician or other health care provider (WHO).

The term “allergic reaction”, as used herein, refers to thehypersensitive response of the immune system of an allergic individualto a substance by an immunoglobulin E (IgE) mediated reaction in theorganism. Such allergic reactions are also known in the art as type Ihypersensitivity reactions. The term allergic reaction or allergicreactions are used broadly to refer to such reactions and to diseases orsymptoms associated with such reactions including allergic rhinitis,allergic asthma, anaphylaxis, wheal and flare reaction, eczema,urticaria and dermatitis.

The term “hyposensibilization”, as used herein, refers to a medicaltreatment that affects the natural course of an allergic disease, andthat may cure allergy. The treatment is by injecting graduallyincreasing doses of the allergen to which the patient is allergic. Byincreasing the dosage very gradually, the immune system may then get theopportunity to “learn” to react correctly when exposed to the allergenin question. The treatment seems to work by inducing the formation of“blocking antibodies” of the IgG4 subclass, which prevent the allergenfrom triggering an allergic reaction. The injection programme usuallycarries on for a period of three to five years. In the initial phase,the dosage is gradually increased from a very low initial value.Typically, the total increase of dosage may by a factor of 1:10000.

In accordance with the above and in relation with the embodiments ofthis invention, the present invention relates also to the use of thepolynucleotide, the polypeptide or the antibody for the preparation of apharmaceutical composition for use in hyposensibilization or in medicalintervention of an allergic reaction. The pharmaceutical composition isparticularly useful in the medical intervention of allergies againstCaviidae.

In a further embodiment, the present invention relates to the use ofsaid polynucleotide, the said polypeptide or the said antibody indiagnosis of allergic reaction or for the determination of a subjectprone to an allergic reaction. Furthermore, the above-mentionedcomponents can be also used for the preparation of a diagnosticcomposition for the diagnosis of allergic reaction or for thedetermination of a subject prone to an allergic reaction. The subject ispreferably a human subject.

The compounds of the present invention (polypeptides and fragmentsthereof, polynucleotides, vectors, host cells, primers etc.) may also becomprised in a diagnostic composition.

The term “diagnostic composition” as herein described, refers to one ofthe aforementioned compounds which is prepared to be used for diagnosticpurposes. It is to be understood that depending on the nature of thediagnostic agent, i.e. depending on whether a polynucleotide, apolypeptide (or a fragment thereof), an antibody or an oligonucleotideis used, the diagnostic composition may comprise additional agents suchas agents which allow hybridization, antibody binding or detection. Suchadditional agents are well known to those skilled in the art.

The term “diagnosis” as used herein means identification of pathologicalcondition and features. For the purpose of the invention, a diagnosis isto identify the allergenicity to a Caviidae allergen(s) of anindividual. One of the known diagnosis method to be used in accordancewith the present invention is the skin test which can be performed byplacing a small amount of a suspected allergen on or below the skin tosee if a reaction develops. However, there are different types of skintests. The skin prick test can be applied by placing a drop of asolution containing a possible allergen on the skin, and a series ofscratches or needle pricks allows the solution to enter the skin. If theskin develops a red, raised itchy area, it usually means that the personis allergic to that allergen indicating a positive reaction. In theintradermal test a small amount of the allergen solution is injectedinto the skin. An intradermal allergy test may be done when a substancedoes not cause a reaction in the skin prick test but is still suspectedas an allergen for that person. The intradermal test is more sensitivethan the skin prick test but is more often positive in people who do nothave symptoms to that allergen (false-positive test results). For a skinpatch test, the allergen solution is placed on a pad that is taped tothe skin for 24 to 72 hours. This test can be used to detect a skinallergy, e.g. contact dermatitis.

Another known method to diagnose allergenicity is the allergy blood testwhich aims at the identification of substances in the blood, e.g.antibodies. Blood tests are not as sensitive as skin tests but are oftenused for people who are not able to have skin tests. A common type ofblood test used is the enzyme-linked immunosorbent assay (ELISA, EIA).It measures the blood level of a type of antibody (called immunoglobulinE or G (IgE/IgG)) that the body may make in response to certainallergens. Other lab testing methods, such as radioallergosorbenttesting (RAST), an immunoassay capture test (ImmunoCAP, UniCAP, orPharmacia CAP), a cytometry beads array (CBA) or a microarray,preferably a protein microarray, may be used to provide moreinformation. Protein microarrays (also protein binding microarray,biochip, proteinchip) are measurement devices used in biomedicalapplications to determine the presence and/or amount (referred to asquantitation) of polypeptides/proteins or fragments thereof inbiological samples, e.g. blood. The protein microarray provides amultiplex approach to identify protein-protein interactions or toidentify the targets of biologically active small molecules. In thiscontext, the polypeptides/proteins or fragments thereof can beexternally synthesised, purified and attached to the array.Alternatively they can be synthesised in-situ and directly attached tothe array. In the present invention, the array can be a piece of glassor silicon on which different molecules of polypeptides/proteins orfragments thereof have been affixed at separate locations in an orderedmanner thus forming a microscopic array. The protein microarray of thepresent invention can also be used as an antibody microarray, whereantibodies are spotted onto the protein chip and are used as capturemolecules to detect proteins in biological samples, e.g. blood.

The appended examples document how the novel polypeptides (or fragmentsthereof) can be used in diagnostic setting. For example, the presentinvention provides also the quantification of specific IgE antibodies to(r)Cavia porcellus allergen 1a, (r)Cav p 2, (r)Cav p 3 and/or (r)Cav p6. Purified recombinant Cavia porcellus allergen 1a, Cav p 2, Cav p 3and Cav p 6 are coated overnight at 4° C. at a concentration of 5 μg/mlin PBS to microtiter plates. Plates are blocked with blocking buffer (3%bovine serum albumin (BSA, Sigma) in TBS/0.05% Tween-20 for Caviaporcellus allergen 1a and 2% cold water fish gelatin (CWFG, Sigma) forCav p 2, 3 and 6) for 30 min at room temperature. Patient sera can beserially diluted into blocking buffer and may be added to the microtiterplates for 2 hours. Binding of IgE is measured by incubation, forexample, for 1 hour with biotin-labeled antihuman IgE antibodies(1/2000); Southern Biotechnology, Birmingham, Ala.) followed by anincubation for 30 min with alkaline phosphatase-labeled streptavidine(1/1000; BD Biosciences Pharmingen, Erembodegem, Belgium). As detectionmeans herein, p-Nitrophenyl phosphate can be added as substrate, andplates were left in the dark before OD was read at 405 nm. The cutoffmay be determined as the mean of the negative controls plus 3 SD. Theserum of a patient with a known titer of specific IgE to cat serumalbumin determined by Phadia CAP-System (Phadia, Uppsala, Sweden) areserially diluted to generate a standard curve on which alldeterminations of specific IgE were determined. Appended Example 6provides for illustrative data in this respect.

Furthermore, nucleic acids, polypeptides/proteins, antibodies of thepresent invention can also be spotted onto solid carriers, like chips.

Taking advantage of the herein provided means and methods a personskilled in the art is easily in the position to diagnose an allergicreaction to a Caviidae allergen or to determine a subject prone to anallergic reaction to Caviidae allergen.

In a further embodiment, the present invention relates to an in vitromethod for determining allergenicity comprising the steps of (a)exposing a sample to the defined polypeptide(s) or antibody; and (b)determining immunological reaction or the formation of an immunologicalcomplex to the exposure of step (a).

In this in vitro method, said immunological reaction to be assayed maycomprise an increased titer of immunglobulines, preferably IgE, IgG4 orIgG1, activation of basophiles, an increased histamine release,leukotriene release, any mediator specifically released by an immunecell in an IgE, IgG4 or IgG1 mediated allergic reaction or whereby saidformation of immunological complex is the formation of anantibody-antigen complex between either said polypeptide and an antibodyor the above-mentioned antibody (like IgE, IgG4 or IgG1) and acorresponding antigen. An increased titer of immunglobulines and/or anincreased histamine release is indicative for an immunological orallergic reaction. It is preferred that the immunological or allergicreaction is a reaction to Caviidae and a preferred immunoglobuline isIgE, IgG4 or IgG1.

The term “formation of an immunological complex” as used herein refersto the formation of an antibody-antigen complex between either thepolypeptide provided herein and an antibody or the antibody and acorresponding antigen. Said immunological or allergic reaction can be areaction to polypeptides/proteins or fragments thereof of Caviidae.

The term “allergenicity” as herein described, refers to the property ofan individual of being allergenic.

In a preferred embodiment, the sample to be used in accordance with thepresent invention may, for example, be a biological sample, a medicalsample or an environmental sample. The environmental sample(s) may beany sample from air or dust or from home or working place, such ascarpet, tissue or filter samples. For instance, the biological ormedical sample comprises cell(s) or tissue(s) taken, e.g. by theattending physician, from an individual. Such a biological sample mayalso be a blood sample, a serum sample and the like. But also skinsample or sputum and mucosal samples may be used. Exemplarily, but notlimiting, the biological or medical sample to be analysed in context ofthe present invention may or may be derived from a patient or a subjectsuspected to suffer from or being prone to suffer from an allergy to (a)Caviidae allergen(s), e.g. skin, respiratory system or mucosa.Exemplarily, but not limiting, the biological or medical sample to beanalysed in context of the present invention is or is derived fromblood, plasma, white blood cells, urine, semen, sputum, cerebrospinalfluid, lymph or lymphatic tissues or cells, amniotic fluid, hair, hairand/or follicles, stem cells (embryonal, neuronal, and/or others) orprimary or immortalized cell lines (lymphocytes, macrophages, or celllines). Alternatively, a sample obtained, for example, from an animal ofthe Caviidae family may be used in the assessment of the allergenicpotential of said animal, e.g. by binding of the herein describedantibodies directed against the novel peptides/polypeptides of thisinvention to the Caviidae allergen.

The use of the herein provided Caviidae allergens/antigens incombination is envisaged in the means and methods of the presentinvention, wherein the combination of the novel Caviidae allergens ofthe present invention (Cav p 3, Cav p 6 or Cavia porcellus allergen 1a)is particularly preferred. However, Cav p 3, Cav p 6 and/or Caviaporcellus allergen 1a (or (a) fragment(s) thereof) may also be used byitself or in combination with known guinea pig allergens like Cav p 1 orCav p 2 (Fahlbusch (2002) loc.cit; Fahlbusch (2003) loc.cit). Theexplanations and definitions given herein above in context of theCaviidae allergens of the present invention, in particular in context ofidentifying variant sequences of the nucleic acid molecules/polypeptidesof the present invention also apply, mutatis mutandis, to known guineapig allergens (e.g. Cav p 1 or Cav p 2) to be used in accordance withthe present invention. The (recombinant) polypeptides or fragmentsthereof may be applied e.g. for the immunization (immunotheapy) byoneself or in combination with each other, e.g. (r)Cav p 1 with (r)Cav p2, (r)Cav p 1 with (r)Cav p 3, (r)Cav p 1 with (r)Cav p 6, (r)Cav p 2with (r)Cav p 3, (r)Cav p 2 with (r)Cav p 6, (r)Cavia porcellus allergen1a with (r)Cav p 1, (r)Cavia porcellus allergen 1a with (r)Cav p 2,(r)Cavia porcellus allergen 1a with (r)Cav p 3, (r)Cavia porcellusallergen 1a with (r)Cav p 6, Cavia porcellus allergen 1a with (r)Cav p1, (r)Cav p 2 and (r)Cav p 3, (r)Cavia porcellus allergen 1a with (r)Cavp 1 and (r)Cav p 2 or (r)Cav p 3, (r)Cav p 3 with (r)Cav p 6 or (r)Cav p2 with (r)Cav p 3 and (r)Cav p 6. As shown in appended Example 6 andExample 8 of the present invention, recombinant polypeptides ((r)Cav p2, (r)Cav p 3, (r)Cav p6, (r)Cavia porcellus allergen 1a) can be usedfor the immunization of mice sera to screen tissues and secretionproducts.

In a further embodiment, the present invention relates to a kitcomprising said vector, said recombinant host cell or the antibody asabove-mentioned. Said kit may also comprise primers of the presentinvention. The polypeptides (or fragments thereof) of the presentinvention may also be comprised in kits. The kits of the presentinvention may be useful in diagnostic settings as well as in medicalinterventions. This kit can be used in diagnosis of allergic reaction orfor the determination of a subject prone to an allergic reaction. Thekit of the present invention may comprise further components, like meansof detection (like secondary antibodies, labeled antibodies). It maycomprise negative or positive control samples. The kit (to be preparedin context) of this invention or the methods and uses of the inventionmay further comprise or be provided with (an) instruction manual(s), forexample how carry out the diagnostic assays and methods provided herein,like the detection methods for allergic responses towards theantigens/allergens provided herein. For example, said instructionmanual(s) may guide the skilled person (how) to use the polypeptides (orfragments thereof of this invention) in the detection of immunoglobulintiters (like IgE-, IgG-titers) in patient samples, in accordance withthe present invention. Particularly, said instruction manual(s) maycomprise guidance to use or apply the herein provided methods or uses.The kit (to be prepared in context) of this invention may furthercomprise substances/chemicals and/or equipment suitable/required forcarrying out the methods and uses of this invention. For example, suchsubstances/chemicals and/or equipment are solvents, diluents and/orbuffers for stabilizing and/or storing (a) compound(s) required for theantigens/allergens provided herein or the polynucleotides encoding thesame or specific antibody molecules directed against the hereindisclosed novel antigens/allergens.

In a further embodiment, the present invention relates to a method foridentifying molecules which are capable of interacting with the saidpolypeptide of comprising (a) producing cells which express saidpolypeptide; (b) contacting the polypeptide produced in step (a) with atest sample potentially containing said molecule; and (c) identifyingamong these molecules the molecules which are capable of interactingwith said polypeptide.

The term “identifying molecules”, as used herein, refers to a method foridentifying a molecule compound, which may be performed by using thecell culture medium or the supernatant thereof into which thepolypeptide has been secreted. Also, the polypeptide may be purified toa degree which is necessary by conventional techniques in order toobtain significant results in said method. In a preferred embodiment,the polypeptide may be used in an immobilized form, i.e. directly orindirectly via the linkage over suitable intermediate molecules (e.g.peptide linkers, antibodies and the like) attached to a solid support.

The term “contacting the polypeptide with a test sample”, as usedherein, refers to a method of evaluating a compound for the ability tointeract with one of the defined polypeptide. The method includes:contacting the compound with the polypeptide; and evaluating the abilityof the compound to interact with, e.g., to bind or form a complex with,the defined polypeptides as shown in Seq ID Nos 2, 4, 6 and 20. Thismethod can be performed in vitro, e.g., in a cell free system, or invivo, e.g., in a two-hybrid interaction trap assay. This method can beused to identify naturally-occurring molecules that interact with theabove-mentioned polypeptides. It can also be used to find natural orsynthetic inhibitors of the above-mentioned polypeptides. In particularembodiments of the invention assays are used wherein the polypeptide isimmobilized on a solid support. A variety of appropriate solid supportsis known to persons of skill in the art. Exemplary supports may includebeads, such as resin beads, slides, glass and plastic wells, test tubes,and plates.

The attachment of the polypeptide to a solid support may facilitateperforming the steps of the methods of the present invention, including,for example, contacting the polypeptide with a compound. Attachment to asolid support will also facilitate washing the polypeptide betweensteps. To a person of skill in the art, it will be apparent that any ofthe above described methods may comprise washing steps between, forexample, the contact of the polypeptide with the compound and contactwith a noxious substance. Further, washing after contacting thepolypeptide with the compound may also be performed before determining,for example, the degree of fragmentation or binding or stability of thepolypeptide. The present invention also encompasses embodimentscomprising at least one or multiple washing steps between at least oneor more of the steps of the above-mentioned methods.

The present invention is further described by reference to the followingnon-limiting figures and examples.

The Figures show:

FIG. 1: Detection of IgE reactive proteins in sub-maxillary glandextract by immunoblot.

FIG. 2: Detection of IgE reactive proteins in harderian gland extract byimmunoblot.

-   -   Remark: Numbers of patient sera do not correspond to the        numbering used in FIGS. 13 and 15.

FIG. 3: 2D-gel of harderian gland extract stained with Coomassie Blue.

FIG. 4: Cav p 2 cDNA nucleotide sequence and deduced amino acidsequence. In the DNA sequence (SEQ ID NO: 5), the corresponding startcodon is at nt 53-55 and stop codon at nt 563-565 is underlined, cDNAcoding for signalpeptide is in italic (nt 53 to 100), cDNA coding formature Cav p 2 is at nt 101-565; in the amino acid sequence (SEQ ID NO:6), the signal peptide is marked up in italic, the determined N-terminalsequence is underlined.

FIG. 5: Analysis of purified (r)Cav p 2 (lane 1), (r)Cav p 3(lane 2) and(r)Cav p 6 (lane 3) by SDS_PAGE and silver staining under denaturing (+)and non-denaturing (-Mercaptoethanol) conditions.

FIG. 6: Detection of Cav p 2 in different guinea pig protein extracts byimmunoblot using a polyclonal mouse anti-(r)Cav p 2 serum.

FIG. 7: 2D gel of submaxillary gland extract stained with CoomassieBlue. Identification of Cav p 3 by N-terminal sequencing

FIG. 8: Cav p 3 cDNA nucleotide sequence and deduced amino acidsequence. In the DNA sequence (SEQ ID NO: 1), the corresponding startcodon is at nt 59-61 and stop codon at nt 569-571 is underlined, cDNAcoding for signalpeptide is in italic (nt 59 to 103), cDNA coding formature Cav p 3 is at nt 104-571; in the amino acid sequence (SEQ ID NO:2), the signal peptide is marked up in italic, the determined N-terminalsequence is underlined.

FIG. 9: Detection of Cav p 3 in different guinea pig protein extracts byimmunoblot using a polyclonal mouse anti-rCav p 3 serum.

FIG. 10: Cav p 6 cDNA nucleotide sequence and deduced amino acidsequence. Amino acids marked up bold represent putative isoforms whichhave been detected by sequencing different cDNA clones.

-   -   In the DNA sequence (SEQ ID NO: 3), the corresponding start        codon is at nt 92-94 and stop codon at nt 635-637 is underlined,        cDNA coding for signalpeptide is in italic (nt 92 to 148), cDNA        coding for mature Cav p 6 is at nt 149-637; in the amino acid        sequence (SEQ ID NO: 4), the signal peptide is marked up in        italic, the determined N-terminal sequence is underlined.

FIG. 11: Detection of Cav p6 in different guinea pig protein extracts byimmunoblot using a polyclonal mouse anti-rCav p 6 serum.

FIG. 12: (r)Cav p 2, (r)Cav p 3 and (r)Cav p 6 are recognized by patientIgE antibodies in immunoblot No 15, 17, 22, 25 and 27 are individualsera of patients allergic to guinea pig.

FIG. 13: IgE reactivity to rCav p 2, rCav p 3 and rCav p 6 assayed byELISA in 27 patients. Values are compared to ImmunoCAP e6 (Phadia,Uppsala, Sweden).

-   -   Remark: Values are cut at 100 kU/I to compare to CAP results.

FIG. 14: IgE reactivity to (r)Cav p 2, (r)Cav p 3 and (r)Cav p 6 assayedby ELISA in 27 patients.

FIG. 15: IgE reactivity of guinea pig allergic patients to recombinantallergens (r)Cav p 2, (r)Cav p 3 and (r)Cav p 6 in comparison toImmunoCAP e6.

FIG. 16: Twodimensional SDS-PAGE gel (20%) of harderian gland extractstained with Coomassie Blue.

FIG. 17: Separation of harderian gland extract by anion exchangechromatography on Resource Q. Harderian gland extract and pooledenriched fractions are analysed by silver stained 15% SDS-PAGE. (M,molecular weight marker; Extr, harderian gland extract; PF, Caviaporcellus allergen 1a enriched fractions).

FIG. 18: Isolation of Cavia porcellus allergen 1a by anion exchangechromatography on Mono Q. Analysis of individual fractions by silverstained 15% SDS-PAGE under denaturing conditions (M, molecular weightmarker; B38-2A5, individual eluted fractions).

FIG. 19: Analysis of the 22 kD allergen under reducing (red) andnon-reducing conditions (oxid). Fractions containing the 22 kD allergenisolated from harderian gland by anion exchange chromatography wereanalysed by 15% SDS-PAGE followed by silver staining.

FIG. 20: Cavia porcellus allergen 1a cDNA nucleotide sequence anddeduced amino acid sequence. In the DNA sequence (SEQ ID NO: 19), thecorresponding start codon is at nt 55-57 and stop codon at nt 553-555 isunderlined, cDNA coding for signalpeptide is in italic (nt 55 to 108),cDNA coding for mature Cavia porcellus allergen 1a is at nt 109-555; inthe amino acid sequence (SEQ ID NO: 20), the signal peptide is marked upin italic, the determined N-terminal sequence is underlined.

FIG. 21: Analysis of rCavia porcellus allergen 1a by 15% SDS-PAGE andsilver staining. Migration under reducing (red) and non-reducingconditions (oxid). M, molecular weight marker.

FIG. 22: IgE binding to Cavia porcellus allergen 1a analysed by ELISA.Specific IgE were determined in a group of 27 patients and 41 controls.Controls are a group of 19 IgE neg patients as well as 22 IgE positivepatients without allergy to mammals. The line marks the cutoff forpositivity as calculated from the average of the IgE neg group plus 3SD.

FIG. 23: IgE reactivity to recombinant Cavia porcellus allergen 1a, Cavp 2, Cav p 3 and Cav p 6 assayed by ELISA in 27 patients. Values arecompared to ImmunoCAP e6 (Phadia, Uppsala, Sweden).

FIG. 24: Specific IgE to recombinant Cavia porcellus allergen 1a, Cav p2, Cav p 3 and Cav p 6 assayed by ELISA. Specific IgE were determined in27 guinea pig allergic patients. Lines mark the cutoff for positivitywhich was determined in 19 IgE negative controls by calculating theaverage of the IgE neg group plus 3 SD.

FIG. 25: Purification of Cav p 2 from hair protein extract. A)Separation of soluble hair proteins by anion exchange chromatography. B)Analysis of individual fractions by 15% SDS-PAGE gel followed by silverstaining. The gel was run under non-reducing conditions. C) Analysis ofindividual eluted fractions by immunoblot. A polyclonal mouse anti-Cav p2 serum was used for detection of Cav p 2.

FIG. 26: Analysis of purified Cav p 2 by 15% SDS-PAGE followed by silverstaining. Migration under reducing (red) and non-reducing (oxid)conditions. M, molecular weight marker.

FIG. 27: Purification of Cav p 3 from submaxillary gland extract. A)Separation of soluble submaxillary gland proteins by anion exchangechromatography. B) Analysis of submaxillary gland extract by 15%SDS-PAGE before loading on the column. The gel was run under reducingconditions and stained with Coomassie Blue. M, molecular weight marker,SM, submaxillary gland extract. C) Analysis of individual fractions by15% SDS-PAGE followed by silver staining. The gel was run undernon-reducing conditions. D) Analysis of individual eluted fractions byimmunoblot. A polyclonal mouse anti-Cav p 3 serum was used for detectionof Cav p 3.

FIG. 28: Purification of Cav p 3 by gel filtration.

-   -   Cav p 3 enriched fractions obtained by anion exchange        chromatography were pooled and purified by gel filtration.

FIG. 29: Analysis of purified Cav p 3 by 15% SDS-PAGE followed by silverstaining. Migration under reducing (red) and non-reducing (oxid)conditions. M, molecular weight marker.

FIG. 30: Purification of Cav p 6 by anion exchange chromatography.

-   -   A) Separation of soluble harderian gland proteins by anion        exchange chromatography. B) Analysis of harderian gland extract        by 15% SDS-PAGE before loading on the column. The gel was run        under reducing conditions and stained with Coomassie Blue. M,        molecular weight marker, H, harderian gland extract. C) Analysis        of individual fractions by 15% SDS-PAGE gel followed by silver        staining. The gel was run under non-reducing conditions.

FIG. 31: Detection of Cavia porcellus allergen 1a in different guineapig protein extracts by immunoblot using an anti-Cavia porcellusallergen 1a serum.

FIG. 32: Analysis of skin prick test solutions from different suppliersby SDS-PAGE and immunoblot. A) Prick test solutions from 4 supplierswere analysed by 15% SDS-PAGE followed by silver (500 ng of samples 1and 2) or Coomassie staining (12 μg of samples 3 and 4); B) Detection ofCavia porcellus allergen 1a in prick test solutions using a polyclonalmouse serum raised against Cavia porcellus allergen 1a, Cav p 2, Cav p3, Cav p 6 and GPA. M, molecular weight marker; 1-4, prick testsolutions from 4 different suppliers.

The following Examples illustrate the invention:

EXAMPLE 1 Material and Methods Source Materials and Extract Preparationof Soluble Proteins:

All extracts were derived from male Dunkin Hartley guinea pigs whichwere killed by intra-cardiac injection of barbiturate solution.

Unwashed hair was clipped off using scissors, cut into pieces of 2 mmand shaken at 4° C. overnight with Phosphate buffered saline containinga mixture of protease inhibitors (Protease Inhibitor Cocktail Tablets,Roche, Mannheim, Germany). The suspension was centrifuged for 30 min at12 000 g at 4° C. to remove hair and solid particles. The supernatantwas concentrated with an Ultrafree concentrator (cut off 5000 MW,Millipore, Bedford, Mass.) and frozen at −20° C.

After hair clipping, the entire skin (extraneous fat was excised) wascollected. The skin was cut into 3 mm pieces and degreased by shakingovernight in diethyl ether (1:10, v:v) at 4° C., dried at roomtemperature and extracted.

All solid source materials was cut into 3 mm pieces, frozen in liquidnitrogen, grinded with mortar and pestle to fine powder and suspended inSingle Detergent Lysis buffer (50 mM TRIS-HCl pH 8, 150 mM NaCl, 1%Triton X-100, protease inhibitors). The extract was centrifuged for 30min at 12 000 xg at 4° C. to remove the insoluble particles. Thesupernatant was concentrated with an Ultrafree concentrator (cut off5000 MW, Millipore).

Serum was collected by intra-cardiac blood puncture. Urine was drawndirectly out the bladder with a needle and syringe.

Salivation was induced with pilocarpine nitrate salt (Sigma, St. Louis,Mo.) (1 ml of a 4% solution in the mouth) before anesthesia with aformulation of ketamine and domitor (subcutaneously injection).

Patient Sera

Sera of patients with allergy to guinea pigs were selected for thisstudy. Selection was based on clinical history, rhinitis and/or asthma,positive skin prick test results to guinea-pig commercial allergenextracts and positive specific IgE results (>17 kU/I) to Caviidae (e6)using CAP system (Phadia, Uppsala, Sweden). Sera of 19 IgE negativepatients were collected as controls for specific IgE determination inELISA. Another control group consisted of 22 IgE positive patientsallergic to pollen and/or mite with total IgE ranging from 16 to 2051kU/L.

SDS-PAGE and Allergen Detection by Specific IgE (Human) or IgG (Mouse)Antibodies in Immunoblot

The different extracts and purified proteins were studied by SDS-PAGEgels under reducing or non-reducing conditions. Proteins were detectedwith Coomassie stain (GelCode Blue Stain reagent, Pierce, Rockford,Ill.) or silver stain (SiverSNAP stain kit II, Pierce). Forimmunoblotting, proteins were transferred onto a PVDF membrane withTRIS-HCl-glycine buffer (25 mM TRIS-HCl, 192 mM glycine, 20% methanol)in semi-dry conditions (14V, 900 mM, 35 min). To reduce non-specificbinding, blotted membranes were incubated in blocking buffer (TRIS-HClbuffered saline/tween-20 0.05%, 2% cold water fish gelatine (Sigma), for2 hours.

PVDF membranes were incubated overnight with patient sera dilutedone-tenth in blocking buffer or mouse anti-sera (1/5000) raised againstguinea pig allergens. Control strips were incubated with buffer alone.Strips were washed three times with TBS/Tween-20 0.05%. Bound IgE weredetected with anti-human IgE antibody (1:1 000) labelled with alkalinephosphatase (Sigma). Bound mouse IgG were detected with anti-mouse IgGantibody (1:10 000) labelled with alkaline phosphatase (Sigma). Blotswere developed by addition of nitro bluetetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) (Promega,Madison, Wis.).

2D-Gels

2D-gels were performed according to the conditions described by themanufacturer (Bio-Rad, Hercules, Calif.). The first dimensionisoelectro-focusing (IEF) was carried out on immobilized pH gradient(IPG) strips, pH 3-10 or 3-6. Extracts (150-450 μg of soluble proteins)were precipitated by TCA/acetone and resuspended in rehydratation buffercontaining 8M urea, 0.5% CHAPS, 10 mM DTT, 0.2% Bio-Lytes 3-10.

Proteins were separated at 4000V and 50 mA for 6 hours according totheir pl. After a first incubation for 15 min in equilibration buffer(375 mM TRIS-HCl—HCl pH8.8, 6M urea, 2% SDS, 20% glycerol and 130 mMDTT) and a second equilibration (15 min) with iodoacetamide in the SDSequilibration buffer (without DTT), the strips were loaded onto aSDS-PAGE for separating the proteins according to their molecularweight. The 2D-gels were stained with GelCode Blue Stain reagent(Pierce) or blotted onto PVDF membrane (Millipore) in order to performan immunoblotting or N-terminal sequencing.

Purification and Analysis of Guinea Pig Hair Proteins by Anion ExchangeChromatography

15 mg of soluble hair proteins were dialysed into 20 mM TRIS-HCl, pH 8(buffer A) and were loaded onto a RESOURCE Q column (1 ml, GEHealthcare, Buckinghamshire, UK). Unbound material was removed bywashing with buffer A. Bound proteins were eluted by a linear gradientof 0-0.5 M NaCl in 20 mM Tris-HCl, pH8 (buffer B). Eluted fractions werefirst analysed by SDS-PAGE followed by silver staining, than byimmunoblotting with patient serum. IgE reactive bands were analysed byN-terminal sequencing.

Purification of Cavia porcellus Allergen 1a by Chromatography

Harderian gland proteins were extracted as described above and dialyzedwith 20 mM TRIS-HCl, pH 8. 2×25 mg of extract was loaded onto a RESOURCEQ column (1 ml, GE Healthcare, Buckinghamshire, UK). Bound proteins wereeluted by a linear gradient of 0-500 mM NaCl in 20 mM TRIS-HCl, pH8.Eluted fractions were first analysed by SDS-PAGE followed by silverstaining. Fractions containing Cavia porcellus allergen 1a were pooledand dialyzed with 20 mM piperazine, pH 5.5 (Sigma). A total of 1.8 mgenriched Cavia porcellus allergen 1a were further separated by ionexchange chromatography (Mono Q 1 ml, GE Healthcare, Buckinghamshire,UK) using a gradient of 0-500 mM NaCl in 20 mM piperazine, pH 5.5.Individual fractions were selected for N-terminal sequencing.

Amino Acid Sequencing

The N-terminal amino acid sequences of purified proteins were determinedby automated Edman degradation on PROCISE 49×HT Protein sequencer(Applied Biosystems, Foster City, Calif.). Liquid samples were desaltedand adsorbed onto a PVDF membrane by the ProSorb column. Alternativelyproteins were separated by SDS-PAGE and transferred onto PVDF membranesby semi-dry transfer using CAPS-buffer (CAPS 10 mM, MeOH 20%). Proteinswere stained on the membrane with Gelcode® Blue (Pierce) overnight.After destaining with methanol 50%, acetic acid 7%, the membrane waswashed for 5-6 h in ultrapure water and dried. Bands of interest werecut out using a clean scalpel and stored at −20° C. until sequencing.

RNA Extraction

RNA in tissues was stabilized in RNA Stabilization Reagent (Qiagen,Hilden, Germany). After 24 h at 4° C., the reagent was removed andsamples were stored at −80° C.

A total of 200 mg of tissue was used for RNA extractions. Fordisruption, the glands were thawed, cut in fine pieces, ground with amortar and pestle. The powder was suspended in the lysis buffer from theRNeasy lipid tissue midi kit (Qiagen). The suspension was homogenized bypassing through a needle of 20-gauge at least 10 times. The RNAextractions were performed according to the protocol and materialsprovided by the RNeasy lipid tissue midi kit (Qiagen). The quality ofmRNA was checked by formaldehyde agarose gel 1%.

Cloning of cDNA Coding for Cav p 2, Cav p 3 and Cav p 6

The cDNA were obtained according to the conditions and materials of BDSMART RACE cDNA amplification kit (BD Biosciences). 3′ and 5′ RACE wasperformed with sub-maxillary and harderian gland total RNA.

Using the degenerated primers, the PCR products were obtained after atouch down from 66° C. to 58° C. for 12 cycles for Cav p 2, from 60° C.to 54° C. for Cav p 3 and from 62° C. to 57° C. for Cav p 6. These firstcycles were followed by 25 cycles with annealing temperatures of 58° C.,53° C. and 57° C. respectively. The PCR products were analysed by 1.5%agarose gel.

PCR products corresponding to the expected molecular weight were clonedinto pCR2.1-topo for sequence analysis. Sequences obtained allowed todesign specific reverse primers for amplification of the 5′ end. Afterfinal sequence analysis, specific primers were used for cloning the openreading frame into the expression vector pQE60 (Qiagen). DNA sequencewas confirmed again by sequencing.

Cloning of cDNA Coding for Cavia porcellus Allergen 1a

The cDNA was obtained according to the conditions and materials of BDSMART RACE cDNA amplification kit (Clontech-Takara, St-Germain-en-Laye,France). 3′ and 5′ RACE was performed with harderian gland total RNA.

12 forward and 8 reverse degenerate primers (table 1 in example 9) weredesigned on the N-terminal amino acid sequence obtained from Caviaporcellus allergen 1a. Using all possible combinations of thesedegenerate primers, PCR products of the expected size of 95 bp wereobtained with primers Cavp-MQ2 and Cavp-MQ7rev.

The 95 bp DNA fragment was cloned into pCR2.1-topo (Invitrogen,Merelbeke, Belgium) for sequence analysis. Sequences obtained allowed todesign specific forward and reverse primers for amplification of the 3′and 5′ end (table 1 in example 9). After final sequence analysis,specific primers were used for cloning the open reading frame into theexpression vector pQE60 (Qiagen). DNA sequence was confirmed again bydouble strand DNA sequencing.

DNA Sequencing

The DNA sequencing was performed by the method of Sanger using a LI-COR4300 DNA analyser (LI-COR Biosciences, Lincoln, Nebr.).

Expression and Purification of Recombinant Cavia porcellus Allergen 1a,Cav p 2, Cav p 3 and Cav p 6

cDNAs coding for mature proteins Cavia porcellus allergen 1a, Cav p 2,Cav p 3 and Cav p 6 were subcloned into pQE-60 (Qiagen) and wereexpressed in E. coli M15 and Rosettagami cells as recombinant proteinswith a C-terminal 6 Histidine tag. The recombinant proteins werepurified by affinity chromatography (HisTrap™ HP, GE Healthcare) undernative conditions according to the instructions of the manufacturer.Recombinant proteins were eluted by imidazol gradient in 20 mM NaH₂PO₄,500 mM NaCl pH 8 and dialyzed with PBS. The purity of the recombinantproteins was analysed by SDS-PAGE. Identity was confirmed by N-terminalsequencing.

Mice Immunisation and Polyclonal Serum Against (r)Cav p 2

The recombinant Cav p 2 protein was mixed with aluminium hydroxide. Twomice were vaccinated by intra-peritoneal injections with 20 μg ofcoupled (r)Cav p 2/injection. The mouse immunization was performedfollowing a program of 38 days (first injection=0 day, firstboost=14^(th) day, second boost=28^(th) day, harvest at day 38). Thesera from both mice were obtained by intra-cardiac blood puncture,incubation of blood for 30 min at room temperature and centrifugation.

Mice Immunisation and Polyclonal Serum Against (r)Cavia porcellusAllergen 1a, (r)Cav p 3, (r)Cav p 6

50 ug of purified recombinant proteins were suspended in adjuvant(complete Freund's for (r)Cav p 3 and aluminium hydroxyde for (r)Cav p 6and for (r)Cavia porcellus allergen 1a). Three mice were vaccinatedintra-peritoneally. 3 boosts at 3 weeks of interval were administeredusing 50 ug of the same proteins in adjuvant (incomplete Freund's for(r)Cav p 3 and aluminium hydroxyde for (r)Cav p 6 and (r) Caviaporcellus allergen 1a). Sera were harvested 3 to 10 days after the lastboost by intra-cardiac blood puncture. Blood was incubated for 30 min atroom temperature and centrifuged.

Quantification of Specific IgE Antibodies to (r)Cavia porcellus Allergen1a, (r)Cav p 2, (r)Cav p 3 and (r)Cav p 6

Purified recombinant Cavia porcellus allergen 1a, Cav p 2, Cav p 3 andCav p 6 were coated overnight at 4° C. at a concentration of 5 μg/ml inPBS to microtiter plates. Plates were blocked with blocking buffer (3%bovine serum albumin (BSA, Sigma) in TBS/0.05% Tween-20 for Caviaporcellus allergen 1a and 2% cold water fish gelatin (CWFG, Sigma) forCav p 2, 3 and 6) for 30 min at room temperature. Patient sera wereserially diluted into blocking buffer and were added to the microtiterplates for 2 hours. Binding of IgE was measured by incubation for 1 hourwith biotin-labeled antihuman IgE antibodies (1/2000); SouthernBiotechnology, Birmingham, Ala.) followed by an incubation for 30 minwith alkaline phosphatase-labeled streptavidine (1/1000; BD BiosciencesPharmingen, Erembodegem, Belgium). p-Nitrophenyl phosphate was added assubstrate, and plates were left in the dark before OD was read at 405nm. The cutoff was determined as the mean of the negative controls plus3 SD. The serum of a patient with a known titer of specific IgE to catserum albumin determined by Phadia CAP-System (Phadia, Uppsala, Sweden)was serially diluted to generate a standard curve on which alldeterminations of specific IgE were determined.

EXAMPLE 2 Screening of Different Tissues and Secretions from Guinea Pig

Screening of different tissues and secretions from guinea pig showed IgEbinding proteins ranging from 6 kDa to 66 kDa. We could confirm thathair, saliva and urine are the most important allergen sources (Swanson(1984), Am Rev Respir Dis 129, 844-849; Walls (1985), Clinical Allergy15, 241-251; Walls (1985), Clinical Allergy 15, 535-546).

In order to characterize the allergens, hair proteins were purifiedusing anion exchange chromatography. IgE immunoreactive fractions werefurther analysed by N-terminal amino acid sequencing. In a next step,protein extracts of different tissues were prepared and they werescreened for allergen expression by IgE immunoblot. The submaxillarygland as well as the harderian gland were found to contain allergens inthe 5-66 kDa range (FIGS. 1 and 2).

EXAMPLE 3 Characterization, Cloning and Expression of Cav p 2

Harderian gland proteins were analysed by 2D gel electrophoresis. IgEimmunoreactive spots were isolated for N-terminal as sequencing (FIG.3). The N-terminal sequence of the protein spot at 17 kD corresponds tothe N-terminal sequence published by Fahlbusch (2003), Allergy 58,629-634 for Cav p 2.

DSIDYSKVPGNWRTI

Backtranslation of the N-terminal as sequence allowed to designdegenerate primers Cav p 2 a-d:

Seq ID No: 7: Cav p 2a: 5′ AAR GTN CCN GGN AAC TGG CG 3′ Seq ID No: 8:Cav p 2b: 5′ AAR GTN CCN GGN AAT TGG CG 3′ Seq ID No: 9: Cav p 2c: 5′AAR GTN CCN GGN AAC TGG AG 3′ Seq ID No: 10: Cav p 2d: 5′AAR GTN CCN GGN AAT TGG AG 3′

A degenerate primer of SEQ ID No: 10 may also comprise a shortersequence starting from AAR to TGG with 18 nucleotides. However, theskilled person is in a position to modify and amend primers and primersequences as shown in SEQ ID No: 10, for example, by elongation and/orshortening of the sequences or distinct nucleotide exchanges. Aparticular primer which is to be used in accordance with this invention,e.g. SEQ ID NOS: 33-40 (Cavp-MQ1 rev to Cavp-MQ8rev).

Such an exchange needs to lead to a complementary strand sequence thatis still capable to specifically hybridize under conditions disclosedherein to its corresponding sequence on the corresponding complementarystrand.

Oligo Cavp 2b yielded a 800 bp product from submaxillary gland extract.Sequence analysis of the clones obtained identified the cDNA as Cav p 2.A specific reverse primer (Seq ID No:11: Cavp2revGTGTTTTCACCAGCGTAGTCCACAG) was designed to amplify the 5′ end using5′RACE. The combination of the 5′ and 3′ amplifications revealed an openreading frame of 512 nucleotides. A hydropathy plot of the deduced assequence revealed a major hydrophobic region from as 1 to 16. Thisregion corresponds to a secretory signal peptide with a predictedcleavage site corresponding to the site determined by N-terminalsequencing. Cleavage of the signal peptide results in a 154 amino acidmature protein with a predicted molecular weight of 17.1 kDa and anisoelectric point of 4.27. No asparagin-linked glycosylation sites weredetected. The official name ‘Allergenic lipocalin from Cavia porcellusCav p 2.0101’ has been assigned to the protein by the I.U.I.S. allergennomenclature sub-committee.

The Cav p 2 protein sequence (FIG. 4) was aligned to the proteindatabases. Sequence alignments clearly identify the protein aslipocalin. It has an identity of 30-42% to different odorant bindingproteins from mouse and rat, 41% to hamster aphrodisin and to a lesserextent (25-30%) to different mammalian urinary proteins.

A cloning experiment using the specific Cav p 2 primers and RNA from theharderian gland yielded a number of clones identical to Cav p 2 clonedfrom submaxillary gland. A screening of a high number of clones showedsome sequence diversity (up to 2% on as level), possibly pointing out tothe existence of different isoforms as already discussed by Fahlbusch(2003), Allergy 58, 629-634.

The cDNA coding for Cav p 2 was inserted into pQE-60 and Cav p 2 wasexpressed in E. coli M15 and Rosettagami cells with a C-terminal 6H istag (FIG. 5, lane 1).

The serum of a mouse immunized with (r)Cav p 2 was used to screenseveral tissues and secretion products. This serum recognized strongprotein bands in hair and harderian gland extracts. Reactivity to salivaand urine is weak in unconcentrated samples. Faint bands are detected insub-maxillary gland and skin (FIG. 6).

EXAMPLE 4 Characterization, Cloning and Expression of Cav p 3

Submaxillary gland extract was used for IgE immunoblotting. Animmunoreactive band at 19 kDa was isolated for N-terminal sequencing.The same extract was analysed by 2D electrophoresis and animmunoreactive spot at a pl of 5.2 was isolated and the N-terminal asdetermined (FIG. 7).

N-terminal sequence analysis showed the following results:

2D-gel: G   Q T L D L P S E I N G Q W/V/G R/G T/A SDS-PAGE (1D):G/H Q T L D L P S E I N G Q W/H/V H   T Published sequence Cav p 1:                S E I N G D W     N   TIALSAD

The N-terminal sequence may also comprise:

2D-gel: G   Q T L D P S E I N G Q W/V/G R/G T/A SDS-PAGE (1D):G/H Q T L D P S E I N G Q W/H/V H   T Published sequence Cav p 1:              S E I N G D W     N   TIALSAD

The following primers are used (amino acid D was changed into Q):

Seq ID No: 12: Cav p 1e: 5′ GAR ATH AAY GGN CAG TGG AAC AC 3′Seq ID No: 13: Cav p 1f: 5′ GAR ATH AAY GGN CAG TGG AAT AC 3′Seq ID No: 14: Cav p 1g: 5′ GAR ATH AAY GGN CAA TGG AAC AC 3′Seq ID No: 15: Cav p 1h: 5′ GAR ATH AAY GGN CAA TGG AAT AC 3′

Oligo Cav p 1h amplified bands in the 700 to 900 bp range fromsubmaxillary gland extract. Sequence analysis of the clones obtainedidentified the cDNA as Cav p 3 (FIG. 8). A specific reverse primer (SeqID NO:16: Cav p 3rev GCC CTT TTC CCC AGC CAC CTC CTG C) was designed toamplify the 5′ end using 5′RACE. The combination of the 5′ and 3′amplifications revealed an open reading frame of 512 nucleotides. Ahydropathy plot of the deduced as sequence showed a major hydrophobicregion from as 1 to 15. This region corresponds to a secretory signalpeptide with a predicted cleavage site corresponding to the sitedetermined by N-terminal sequencing. Cleavage of the signal peptideresults in a 155 amino acid mature protein with a predicted molecularweight of 17.5 kDa and an isoelectric point of 5.15. No asparagin-linkedglycosylation sites were detected.

The translated cDNA sequence clearly shows that Cav p 3 is differentfrom the protein called Cav p 1 by Fahlbusch (2002), Allergy 57,417-422; Fahlbusch (2003), Allergy 58, 629-634. The N-terminus as wellas 5 as are different from the published 15 aa. The official name‘Allergenic lipocalin from Cavia porcellus Cav p 3.0101’ has beenassigned to the protein by the I.U.I.S. allergen nomenclaturesub-committee.

Cav p 3 HQTLDPSEINGQWHTISIAAD Cav p 1       SEINGDWNTIALSAD

The Cav p 3 protein sequence was aligned to the protein databases.Sequence alignments clearly identify the protein as lipocalin. It has anidentity of 46% to a rat odorant binding protein, 43% to hamsteraphrodisin, 42% to a lacrimal protein of hamster, 40% to the cattleallergen Bos d 2 and 30-45% identity to other mammalian odorant bindingproteins, 20-30% to rodent urinary proteins. The cDNA coding for Cav p 3was inserted into pQE-60 and Cav p 3 was expressed in E. coliRosettagami cells with a C-terminal 6H is tag (FIG. 5, lane 2).

The serum of a mouse immunized with (r)Cav p 3 was used to screenseveral tissues and secretion products (FIG. 9). This serum recognized astrong band at 18-19 kDa in saliva, hair, and submaxillary gland. Cav p3 is not expressed in the harderian gland, in the skin and in foot pads.

EXAMPLE 5 Characterization, Cloning and Expression of Cav p 6

A guinea pig hair extract was purified by anion exchange chromatography.Fractions were analysed by immunoblotting with patient sera and mouseanti-Cav p 2 and anti-Cav p 3 sera. An IgE reactive band at about 19 kDawas detected in several fractions negative for Cav p 2 and Cav p 3. Thisband was subjected to N-terminal sequencing.

hair extract DEVLYGNFDAEKISG harderian gland extract DEVVRGNFDAEKISGN

The same protein could be identified in harderian gland extract, howeverwith 2 as differences. As the protein sequence obtained from hair wasambiguous in several positions, these differences might be due tocontaminants.

Protein alignments with the database showed high similarities betweenour N-terminus and Fel d 4, Equ c 1, alpha-uteroglobulin of the dog, amouse urinary protein and a salivary lipocalin of the pig. DNA sequencesof these proteins were also highly conserved. A degenerated primer Cav p6 was designed which took into account the protein sequence of Cav p 6and the conserved DNA sequences of the homologous proteins.

Seq ID NO: 17: Cav p 6c: GAR AAG ATT TCR GGV AAY TGG TAT

Oligo Cav p 6 amplified a band at about 750 bp from harderian glandextract. Sequence analysis of the clones obtained identified the cDNA asCav p 6 (FIG. 10). A specific reverse primer (Seq ID NO:18: Cav p6_(—)5RACE-2 CTG GTT CTC GGC CAT AGA GCT C) was designed to amplify the5′end using 5′RACE. The combination of the 5′ and 3′ amplificationsrevealed an open reading frame of 545 nucleotides. A hydropathy plot ofthe deduced as sequence revealed a major hydrophobic region from as 1 to15. This region corresponds to a secretory signal peptide with 3different possible predicted cleavage sites at as positions 16, 17 or19. Position 19 corresponds to the site determined by N-terminalsequencing. Cleavage of the signal peptide results in a 162 amino acidmature protein with a predicted molecular weight of 18.5 kDa and anisoelectric point of 4.67. No asparagin-linked glycosylation sites weredetected.

The Cav p 6 protein sequence was aligned to the protein databases.Sequence alignments clearly identify the protein as lipocalin. It has anidentity of 51% to rat alpha-uteroglobulin, 49% to Equ c 1, 42% to Fel d4 and 45-50% to rodent urinary proteins. The high identity to Fel d 4and Equ c 1 is not surprising as already the N-terminal sequence alignedon these proteins.

A total of 77 clones arising from 7 independent PCR's and 2 reversetranscriptions have been sequenced. Different clones with singlemutations as well as a clone having 6 mutations have been isolated. 4 assubstitutions were present in different RT-PCR clonings, suggesting theexistence of at least 5 different Cav p 6 isoforms. A “K to E”substitution at position 47 of the mature protein has been repeatedlyfound in different clones from independent RT-PCR's.

The cDNA coding for Cav p 6 was inserted into pQE-60 and Cav p 6 wasexpressed in E. coliRosettagami cells with a C-terminal 6H is tag (FIG.5, lane 3). Under non-denaturing conditions, rCav p 6 migrates as adouble band.

The serum of a mouse immunized with (r)Cav p 6 was used to screenseveral tissues and secretion products (FIG. 11). This serum detected astrong band at about 18 kDa in saliva, urine, hair, submaxillary gland,skin and foot pads. Bands of 18 and 25 kDa were detected in theharderian gland.

EXAMPLE 6 Allergenicity of (r)Cav p 2, (r)Cav p 3 and (r)Cav p 6

Recombinant proteins were expressed in Rosettagami and purified byaffinity chromatography. Protein purity was assessed by N-terminalsequencing and by SDS-PAGE followed by silver staining (FIG. 5).Purified (r)Cav p 2, (r)Cav p 3 and (r)Cav p 6 were tested for IgEbinding in immunoblot using sera of patients allergic to guinea pig. Therecombinant proteins were shown to be immunoreactive in several patients(FIG. 12).

In immunoblot, proteins are subjected to SDS-PAGE and their conformationdoes not reflect their native structure. Therefore, a series of 27patients with allergy to guinea pig was analysed by ELISA for IgEreactivity to (r)Cav p 2, (r)Cav p 3 and (r)Cav p 6 (FIG. 15).

FIGS. 13 and 14 show the graphical display of the results of the 27patients. 19 out of 27 patients (70.4%) had IgE antibodies to one orseveral of the 3 recombinant proteins. For a number of patients, the sumof the specific IgE values to Cav p 2, Cav p 3 and Cav p 6 correspondsto the e6 CAP value. For others, and especially the ones negative forCav p 2, 3 or 6, the value is below the CAP value, suggesting that atleast one other important IgE reactive protein is still missing for a100% sensitivity of the clinical test.

IgE profiles differ individually. 63% of the patients react to (r)Cav p2, 52% react to (r)Cav p 3 and 63% react to (r)Cav p 6.33% of thepatients react to all 3 allergens. Some patients react only orpredominantly to one allergen (Cav p 2: patients 4, 7, 11; Cav p 6:patients 10, 19, 27). All patients, which were negative for the 3allergens, were tested for IgE reactivity to guinea pig serum albumin(GPA). One of them was clearly positive (no 13), two others were weaklypositive (No 3, 26).

GPA is commercially available. It could be added to the other proteinsfor diagnosis as recombinant or native molecule. It is known from ourstudies with cat allergic patients and from other studies (Hilger(1997), Allergy 52, 179-187; Spitzauer (1995), J Allergy Olin Immunol96, 951-959; Goubran Botros (1996), Immunology 88(3), 340-347) thatabout 22 to 35% of patients allergic to mammalians react to serumalbumins. Most of these recognize also other allergens of these animals,but in rare cases, a monosensitization to serum albumin can occur. Serumalbumin is known as a cross-reactive mammalian allergen. Cat allergicpatients with IgE reactivity to cat serum albumin will probably alsoreact to GPA and this will result in a positive e6 ImmunoCAP without thepatient being truely allergic to guinea pig. The use of the recombinantallergens Cav p 2, Cav p 3 and Cav p 6 will help to distinguish thesepatients from truely guinea pig allergic patients.

The first commercial microarray has been introduced in the clinic forIgE diagnosis. Large scale epidemiological studies are still needed toevaluate the diagnostic sensitivity and specificity of each of therecombinants used in this system. However for a number of allergens, ithas become clear that a single recombinant will not be able to replacethe natural extract. For each allergenic source, we will have to rely onseveral purified native or recombinant molecules.

For other allergens (e.g. birch and grass pollen) it has been shown thatindividual patients present different IgE reactivity profiles. Diagnosisat the molecular level allows the prediction of cross-reactivities andallows the orientation of the patient to avoidance of specific allergensources. Another goal of the component-resolved diagnosis is to enable apatient-tailored immunotherapy.

Today, diagnosis of allergy to guinea pig is done with whole extracts.These however can not be used in microarray systems. Purified natural orrecombinant molecules have to be used in these high throughput systems.The next years will bring a general change from whole extracts to puremolecules.

EXAMPLE 7 Protein Alignments of Cav p 2, Cav p 3 and Cav p 6

Protein sequences of the 3 allergens were aligned to analyse asidentities:

Cav p 2 and Cav p 3 share the highest number of amino acid identities.The GXW motif, signature of the lipocalin family, is present near theN-terminus of all three proteins.

EXAMPLE 8 Protein Purification and Characterization of Cavia porcellusAllergen 1a

Harderian gland proteins were analysed by 2D gel electrophoresis (FIG.16). IgE immunoreactive spots were isolated for N-terminal amino acidsequencing.

The N-terminal sequence of the 19 kD spot with a pl of 4.1 was S E/Q IN/S G D W N/D T I A L S A D.

Further N-terminal sequence analyses were performed on proteins purifiedby anion exchange chromatography from hair and harderian gland. Theresult was always a mixture of two sequences at positions 2, 4 and 8.

In a first step, we designed degenerate forward primers from this aminoacid sequence to amplify the corresponding cDNA by 3′RACE. Amplificationexperiments yielded bands in the 600-1000 bp range from the harderiangland. These amplificates were cloned and analysed by DNA sequencing.However all clones contained inserts representing cDNA unrelated to theallergen sequence.

In a second step, the N-terminal sequencing was extended up to asposition 23. as 9-23 could be read without ambiguity and degenerateprimers were designed on this sequence (ADNKEKIEEG). A number of 3′RACEwere performed and amplified bands were cloned. None of the clonescorresponded to the protein sequence determined by the N-terminalsequencing. Apparently the primers used in PCR had bound to unrelatedmRNAs containing part of the sequence motif used for amplification.

The approach using 3′RACE was not successful and we decided to amplify ashort internal cDNA fragment using 2 specific primers instead of using 1specific and 1 oligo dT. For this purpose however we needed to sequenceup to 40 residues of the allergen. The purification method was improvedby adding a second chromatography step using a high resolution matrix.

Cavia porcellus allergen 1a was purified from 50 mg harderian glandprotein extract using 2 sequential anion exchange chromatographies. Theextract was first loaded on a Resource Q column and Cavia porcellusallergen 1a was eluted by a salt gradient in a TRIS-buffer of pH 8.0(FIG. 17). Fractions containing Cavia porcellus allergen 1a were pooled,dialysed with piperazine pH 5.5 and 1.8 mg of protein was loaded on aMono Q column. The elution profile showed a number of peaks (FIG. 18).

The allergen was found to migrate at a molecular weight of 22 kD underreducing conditions in 15% SDS-gels. However, under non-reducingconditions, the 22 kD band splits into a 15 kD, a 6 kD and a 7 kD band(FIG. 19). These different forms eluted as a broad peak between 105 mMNaCl and 170 mM NaCl from the Mono Q column.

N-terminal sequencing of the different bands gave the following results:

Fraction D9 7 kD protein: S Q I S G D W D T I A L S A DFraction D37 15 kD protein: S E I N G D W N T I A L S A DFraction 2A5 7 kD protein S Q I S G D W D T I A L S Fraction C25 6 +7 kD protein: S Q/E I S/N G D W D/N T I A L S A D

As the 6 kD protein could not isolated by chromatography, a mixture ofthe 6 and 7 kD proteins (fraction C25) was analysed by N-terminalsequencing. At 3 positions, there was a mixture of 2 amino acid peaks ofhigh intensity. When substracting the 7 kD sequence determined forfraction D9, the sequence of the 6 kD protein should be SEINGDWNTIALSAD.The N-terminal sequence of the 6 kD band seems therefore identical tothe sequence of the 15 kD protein. The 15 first amino acids of the 15 kDprotein are identical to the Cav p 1 allergen described by Fahlbusch etal. (2002). The 7 kD protein however is a new allergen called Caviaporcellus allergen 1a. N-terminal amino acid sequencing was run for amaximum number of cycles and a large amino acid sequence could bedetermined, ‘x’ representing unresolved residues:

SQISGDWDTIALSADNKEKIEEGGPLxxYFRQIDDNADDS

The serum of a mouse immunized with recombinant Cavia porcellus allergen1a (produced as in example 10) was used to screen a number of tissuesand secretion products. Extracts were analysed by 15% SDS-PAGE underreducing conditions. Strong bands were detected at 19 kD in theharderian gland and in guinea pig hair extract. Cavia porcellus allergen1a was not detected in urine, saliva, submaxillary gland, liver or footpads (FIG. 31).

EXAMPLE 9 cDNA Cloning of Cavia porcellus Allergen 1a

All attempts to clone Cavia porcellus allergen 1a using a specificforward primer and 3′RACE had failed. We choose to design anotherexperimental setup. The long amino acid sequence obtained in example 8was used to make degenerate forward (aa 5-11) and reverse (aa 30-36)primers in order to amplify a small cDNA fragment corresponding to theobtained N-terminal sequence. The primers were designed in a specialway: the 5 last bases at the 3′ end should not contain ambiguities andthe overall sequence complexity should not exceed 32 possible sequencesper primer (Table 1). Using all possible primer combinations, a total of96 PCRs were performed and analysed by high resolution metaphor agarosegels for the presence of a 95 bp band amplified specifically by the usedprimers.

Primers Cavp-MQ2 and Cavp-MQ7rev amplified a 95 bp DNA fragment by PCRwhich was cloned for sequence analysis. The translated sequencecorresponded to the Cavia porcellus allergen 1a amino acid sequence5-36. A specific forward primer (Cavp-11_for) was designed from thissequence to obtain the 3′ end of the cDNA. Analysis of the 3′ endenabled the choice of a specific primer (Cavp-b_rev) for amplificationof the 5′ end by 5′RACE PCR.

TABLE 1 Primers designed for amplification of a cDNA fragment codingfor the N- terminus of Cavia porcellus allergen 1a SEQ ID No.:Primer name Primer sequence 5′ -> 3′ 21 Cavp-MQ1GGN GAY TGG RAC ACR ATC GC 22 Cavp-MQ2 GGN GAY TGG RAC ACR ATT GC 23Cavp-MQ3 GGN GAY TGG RAC ACR ATA GC 24 Cavp-MQ4GGN GAY TGG RAC ACY ATC GC 25 Cavp-MQ5 GGN GAY TGG RAC ACY ATT GC 26Cavp-MQ6 GGN GAY TGG RAC ACY ATA GC 27 Cavp-MQ7GGN GAY TGG RAT ACR ATC GC 28 Cavp-MQ8 GGN GAY TGG RAT ACR ATT GC 29Cavp-MQ9 GGN GAY TGG RAT ACR ATA GC 30 Cavp-MQ10GGN GAY TGG RAT ACY ATC GC 31 Cavp-MQ11 GGN GAY TGG RAT ACY ATT GC 32Cavp-MQ12 GGN GAY TGG RAT ACY ATA GC 33 Cavp-MQ1revTT RTC RTC DAT YTG TCT AAA 34 Cavp-MQ2rev TT RTC RTC DAT YTG CCT AAA 35Cavp-MQ3rev TT RTC RTC DAT YTG TCT GAA 36 Cavp-MQ4revTT RTC RTC DAT YTG CCT GAA 37 Cavp-MQ5rev TT RTC RTC DAT YTG RCG AAA 38Cavp-MQ6rev TT RTC RTC DAT YTG YCG AAA 39 Cavp-MQ7revTT RTC RTC DAT YTG RCG GAA 40 Cavp-MQ8rev TT RTC RTC DAT YTG YCG GAA 41Cavp-11_for (3′RACE) CTCTGTCTGC TGACAACAAA GAGRAGATCG AAGAGG 42Cavp-b_rev (5′RACE) CGTTTCTTCG GGTGTCAGAG AGTCTC

Combination of the 5′ and 3′ cDNA ends revealed an open reading frame of498 nucleotides (FIG. 20). Sequence analysis software SignalSeqpredicted a secretory signal peptide of 18 amino acids with a cleavagesite corresponding to the site determined by N-terminal sequencing. Themature protein has a predicted molecular weight of 16.4 kD and anisoelectric point of 4.03. No asparagin-linked glycosylation sites weredetected. The deduced amino acid sequence deviates at residues Cys⁵³ andCys⁵⁷ from the protein sequence determined by Edman degradation.Cysteins can not be detected by this method and positions have beenmisinterpreted as aspartic acid.

Cavia porcellus allergen 1a was aligned to protein databases. It isclassified into the lipocalin family and more specifically to theodorant binding protein family. It has an identity of 45% to hamsteraphrodisin and identities to different mammalian odorant bindingproteins ranging from 31% to 39%. Identity to Bos d 2 (cattle danderallergen 2) is 36%.

EXAMPLE 10 Cavia Porcellus Allergen 1a: Production of RecombinantProtein and Determination of IgE Reactivity

The cDNA coding for Cavia porcellus allergen 1a was inserted into pQE-60and the protein was expressed in E. coli Rosettagami cells with aC-terminal hexahistidine tag. Recombinant protein was purified byaffinity chromatography. Purity was assessed by N-terminal sequencingand by SDS-PAGE followed by silver staining (FIG. 21).

The theoretical molecular weight of recombinant Cavia porcellus allergen1a including the hexahistidine tag is 17.584 kD. The apparent molecularweight in 15% SDS-PAGE under reducing conditions is higher. Running theprotein sample under non-reducing conditions induces a mobility shiftindicating that the 4 cysteins of the protein may form 1 or 2 disulfidbridges. A similar mobility shift is observed for the native protein(FIG. 18, lane D9).

IgE reactivity of recombinant Cavia porcellus allergen 1a was analysedby ELISA in a series of 27 guinea pig allergic patients and 19 IgEnegative controls. A second control group consisted of 22 IgE positivepatients without allergy to mammals, but with specific IgE to pollen orhouse dust mite (total IgE range 16-2051 kU/L).

The cutoff value 1.9 kU/L was calculated from the IgE negative patientgroup as the average plus 3 standard deviations. 18 out of 27 patients(67%) had specific IgE antibodies to recombinant Cavia porcellusallergen 1a (FIG. 22).

EXAMPLE 11 IgE Reactivity of (r)Cavia porcellus Allergen 1a, (r)Cav p 2,(r)Cav p 3 and (r)Cav p 6

A series of 27 patients with allergy to guinea pig was analysed by ELISAfor IgE reactivity to (r)Cavia porcellus allergen 1a, (r)Cav p 2, (r)Cavp 3 and (r)Cav p 6.

FIGS. 23 and 24 show the graphical display of the results of the 27patients. 24 out of 27 patients (88.9%) had IgE antibodies to one orseveral of the 3 recombinant proteins. For a number of patients, the sumof the specific IgE values to Cavia porcellus allergen 1a, Cav p 2, Cavp 3 and Cav p 6 corresponds to the e6 CAP value. For others, the valueobtained by ELISA is below the CAP value. There are several possibleexplanations to this: the capacity of antigen binding of our ELISA plateis much lower than that of the CAP system and the binding sites on theELISA plate might have been saturated; another possibility is that atleast one other important IgE reactive protein might still be missing.

IgE profiles differ individually. 67% of the patients react to (r)Caviaporcellus allergen 1a, 63% react to (r)Cav p 2, 52% react to (r)Cav p 3and 63% react to (r)Cav p 6. 33% of the patients react to all 4allergens. Some patients react only or predominantly to one allergen(Cavia porcellus allergen 1a: patients 3, 4, 22, 23; Cav p 2: patients4, 7, 11; Cav p 6: patients 10, 19, 27).

All patients, which were negative for the 4 allergens (no 8, 15 and 26),were tested for IgE reactivity to guinea pig serum albumin (GPA) inELISA. Sera no 2 and 13 were included as they were only weakly reactingto one allergen. Sera no 8, 13 and 26 had specific IgE antibodies to GPA(1.7, 11.3 and 1.4 kU/L), no 15 was weakly positive (0.8 kU/L). Serum no26 has also IgE antibodies to cat and dog dander and to cat serumalbumin (9.3 kU/L). It might be possible that the patient is primarilysensitized to cat and/or dog and has a cross-sensitization to guinea pigbecause of IgE cross-reactivity to GRA.

Serum albumin is known as a cross-reactive mammalian allergen. Catallergic patients with IgE reactivity to cat serum albumin will probablyalso react to GPA and this will result in a positive e6 ImmunoCAPwithout the patient being truly allergic to guinea pig. The use of therecombinant allergens Cavia porcellus allergen 1a, Cav p 2, Cav p 3 andCav p 6 will help to distinguish these patients from truely guinea pig.

Protein sequences of the 4 allergens were aligned to Cavia porcellusallergen 1a to analyse amino acid identities:

EXAMPLE 12 Protein Purification and Characterization of Cav p 2

Purification of Cav p 2 from Hair

Proteins were extracted from hair as described above and dialysed with20 mM TRIS-HCl pH8 using Ultrafree concentrators with a cut off of 3000Da (Millipore). The soluble proteins were filtered through a 0.22 μmfilter and loaded onto a Resource Q column (1 ml, GE Healthcare). Boundproteins were eluted by a linear gradient of 0-500 mM NaCl in 20 mMTRIS-HCl, pH8. Eluted fractions were analysed by SDS-PAGE followed bysilver staining and immunoblotting with mouse anti-Cav p 2 serum.Fractions containing Cav p 2 were pooled and concentrated usingUltrafree concentrators with a cut off of 3000 Da (Millipore).

Result

Cav p 2 is abundant on hair. The protein was purified by anion exchangechromatography using protein extract from hair. 2×5 mg were separated bya 1 ml Resource Q column using a linear gradient of 0-500 mM NaCl in 20mM TRIS-HCl, pH 8. Eluted fractions were analysed by SDS-PAGE followedby silver staining (FIG. 25).

Fractions containing proteins of a molecular weight of 17 kD wereimmunoblotted using a mouse anti-Cav p 2 serum. Cav p 2 eluted within abroad peak at a NaCl concentration of 115-150 mM. A total of 0.6 mg Cavp 2 was obtained from 10.3 mg guinea pig hair proteins. This representsa final yield of 5.8% of soluble hair proteins.

Purified Cav p 2 was analysed by SDS-PAGE (FIG. 26). The protein has anapparent molecular weight of 17 kDa under reducing conditions.Non-reducing conditions induce a mobility shift indicating that the 4cysteins present in the molecule may form one or 2 disulfid bridges.Identity of the purified protein was confirmed by N-terminal sequencingand by immunoblotting using a mouse anti-Cav p 2 serum.

EXAMPLE 13 Protein Purification and Characterization of Cav p3

Purification of Cav p 3 from Submaxillary Gland Extract

Submaxillary gland was extracted as described above and precipitatedwith methanol. 50 mg of precipitated extract were suspended in 20 mMTRIS-HCl pH8. The sample was centrifuged at high speed and the solubleproteins were filtered by a 0.22 μm filter and loaded onto a Resource Qcolumn (1 ml, GE Healthcare). Bound proteins were eluted by a lineargradient of 0-500 mM NaCl in 20 mM TRIS-HCl, pH8. Eluted fractions wereanalysed by SDS-PAGE followed by silver staining and immunoblotting withmouse anti-Cav p 3 serum.

Fractions containing Cav p 3 were pooled and concentrated usingUltrafree concentrators with a cut off of 3000 Da (Millipore). Theconcentrated sample was than loaded on a prepacked gelfiltration column(Superdex 75, 10/300 GL, GE Healthcare) and proteins were eluted in 50mM KH₂PO₄, 150 mM NaCl, pH7. Fractions were analysed by SDS-PAGEfollowed by silver staining and fractions containing Cav p 3 werecombined, concentrated and dialysed into PBS.

Result

Cav p 3 was found to be abundant in the submaxillary gland. The proteinwas purified by anion exchange chromatography using protein extract fromthe submaxillary gland. A total of 50 mg of extract were separated by a1 ml Resource Q column using a linear gradient of 0-500 mM NaCl in 20 mMTRIS-HCl, pH 8. Eluted fractions were analysed by SDS-PAGE followed bysilver staining and immunoblot (FIG. 27).

Fractions of all peaks were analysed by SDS-PAGE and fractionscontaining proteins of a molecular weight of 18-20 kD were immunoblottedusing a mouse anti-Cav p 3 serum. Cav p 3 eluted as a single peak at aNaCl concentration of 105-130 mM. Analysis of the pooled fractionsshowed that Cav p 3 was contaminated with a 66 kD protein and Cav p 3was further purified by gel filtration (FIG. 28) using Superdex 75resin.

The gel filtration could not totally separate the two proteins.Fractions A29 to B10 were pooled and analysed by SDS-PAGE (FIG. 29).Silver staining showed that Cav p 3 is still contaminated by the 66 kDprotein. The 66 kD protein was suspected to be guinea pig serum albumin.This could be confirmed by immunoblot using an anti-GPA mouse serum.

Under reducing conditions, Cav p 3 runs at an apparent molecular weightof 20 kD. Non-reducing conditions induce a mobility shift indicatingthat the 4 cysteins present in the molecule may form one or 2 disulfidbridges. Identity of Cav p 3 was confirmed by immunoblot using aanti-Cav p 3 mouse serum.

EXAMPLE 14 Protein Purification and Characterization of Cav p6

Purification of Cav p 6 from Harderian Gland Extract

Harderian gland was extracted as described above and dialysed in 20 mMTRIS-HCl pH8. The sample was filtered by a 0.22 μm filter and 20 mg ofprotein were loaded onto a Resource Q column (1 ml, GE Healthcare).Bound proteins were eluted by a linear gradient of 0-500 mM NaCl in 20mM TRIS-HCl, pH8. Fractions were analysed by 15% SDS-PAGE followed bysilver staining.

Result

Cav p 6 was identified on hair and in harderian gland extract. Cav p 6was purified from the harderian gland by anion exchange chromatography.A total of 20 mg of extract were separated by a 1 ml Resource Q columnusing a linear gradient of 0-500 mM NaCl in 20 mM TRIS-HCl, pH 8. Elutedfractions were analysed by SDS-PAGE followed by silver staining andimmunoblot (FIG. 30).

Cav p 6 had initially been identified in a purification of hair extractby N-terminal sequencing. The IgE reactive protein eluted directly afterCav p 2 in a NaCl gradient. Therefore individual fractions were analysedwith mouse anti-Cav p 2 serum. Cav p 2 was found to be present infractions 1030 to 1D35. The dominant 20 kD band in fraction 2A5 wasanalysed by N-terminal sequencing and identified as Cav p 6.

N-terminal sequence: DEVVRGNFDAEKISGN

Cav p 6 is present in fractions 2A4 through 2A15. It elutes as a smallpeak at a concentration of 150 mM NaCl.

EXAMPLE 15 Detection of Guinea Pig Allergens in Commercial Skin PrickTest Solutions

The protein content of skin prick test solutions from 4 suppliers wasquantified. Protein quantity was highly variable ranging from 0.13 mg/mlto 1.6 mg/ml. A sample of each prick test solution was analysed by 15%SDS-PAGE gel. Due to low protein concentrations, samples 1 and 2 werestained with silver, samples 3 and 4 with Coomassie (FIG. 32 A).

Polyclonal mouse sera raised against Cavia porcellus allergen 1a, Cav p2, Cav p 3, Cav p 6 and GPA were used for detection of allergens byimmunoblot. GPA was detected in all 4 extracts. Cavia porcellus allergen1a was detected in extracts 2, 3 and 4. Cav p 6 was detected in extracts2 and 4. Weak bands were visualized by anti-Cav p 2 and anti-Cavp 3 serain extract 4.

These results highlight the value of purified and/or recombinantmolecules. Antibodies can be raised against single components and usedfor standardization of whole extracts. Single molecules can be added toextracts to compensate for insufficient allergen quantity.

The protein content as well as the allergen content is highly variablein commercial skin prick test solutions. The set up of tools forstandardization of animal extracts is mandatory to improve the qualityof those extracts.

The present invention refers to the following nucleotide and amino acidsequences:

SEQ ID No. 1:

Nucleotide sequence encoding Caviidae allergen Cav p 3 (cDNA).

SEQ ID No. 2:

Amino acid sequence of the Caviidae allergen Cav p 3.

SEQ ID No. 3:

Nucleotide sequence encoding Caviidae allergen Cav p 6 (cDNA).

SEQ ID No. 4:

Amino acid sequence of the Caviidae allergen Cav p 6.

SEQ ID No. 5:

Nucleotide sequence encoding Caviidae allergen Cav p 2 (cDNA).

SEQ ID No. 6:

Amino acid sequence of the Caviidae allergen Cav p 2. (Accession Numberof Swissprot: P83508)

SEQ ID No. 7:

Nucleotide sequence of degenerate Cav p 2a primer used for amplificationof the Cav p 2 gene.

SEQ ID No. 8:

Nucleotide sequence of degenerate Cav p 2b primer used for amplificationof the Cav p 2 gene.

SEQ ID No. 9:

Nucleotide sequence of degenerate Cav p 2c primer used for amplificationof the Cav p 2 gene.

SEQ ID No. 10:

Nucleotide sequence of degenerate Cav p 2d primer used for amplificationof the Cav p 2 gene.

SEQ ID No. 11:

Nucleotide sequence of degenerate Cav p 2rev primer used foramplification of the 5′ end of the Cav p 2 gene.

SEQ ID No. 12:

Nucleotide sequence of degenerate Cav p 1e primer used for amplificationof the Cav p3 gene.

SEQ ID No. 13:

Nucleotide sequence of degenerate Cav p if primer used for amplificationof the Cav p3 gene.

SEQ ID No. 14:

Nucleotide sequence of degenerate Cav p 1g primer used for amplificationof the Cav p 3 gene.

SEQ ID No. 15:

Nucleotide sequence of degenerate Cav p 1 h primer used foramplification of the Cav p 3 gene.

SEQ ID No. 16:

Nucleotide sequence of degenerate Cav p 3 rev primer used foramplification of the Cav p 3 gene.

SEQ ID No. 17:

Nucleotide sequence of degenerate Cav p 6 primer used for amplificationof the Cav p 6 gene.

SEQ ID No. 18:

Nucleotide sequence of degenerate Cav p 6_(—)5RACE2 primer used foramplification of the 5′ end of the Cav p 6 gene.

SEQ ID No. 19:

Nucleotide sequence encoding Cavia porcellus allergen 1a (cDNA).

SEQ ID No. 20:

Amino acid sequence of the Cavia porcellus allergen 1a.

SEQ ID No. 21:

Nucleotide sequence of degenerate Cavp-MQ1 primer used for amplificationof the Cavia porcellus allergen 1a gene.

SEQ ID No. 22:

Nucleotide sequence of degenerate Cavp-MQ2 primer used for amplificationof the Cavia porcellus allergen 1a gene.

SEQ ID No. 23:

Nucleotide sequence of degenerate Cavp-MQ3 primer used for amplificationof the Cavia porcellus allergen 1a gene.

SEQ ID No. 24:

Nucleotide sequence of degenerate Cavp-MQ4 primer used for amplificationof the Cavia porcellus allergen 1a gene.

SEQ ID No. 25:

Nucleotide sequence of degenerate Cavp-MQ5 primer used for amplificationof the Cavia porcellus allergen 1a gene.

SEQ ID No. 26:

Nucleotide sequence of degenerate Cavp-MQ6 primer used for amplificationof the Cavia porcellus allergen 1a gene.

SEQ ID No. 27:

Nucleotide sequence of degenerate Cavp-MQ7 primer used for amplificationof the Cavia porcellus allergen 1a gene.

SEQ ID No. 28:

Nucleotide sequence of degenerate Cavp-MQ8 primer used for amplificationof the Cavia porcellus allergen 1a gene.

SEQ ID No. 29:

Nucleotide sequence of degenerate Cavp-MQ9 primer used for amplificationof the Cavia porcellus allergen 1a gene.

SEQ ID No. 30:

Nucleotide sequence of degenerate Cavp-MQ10 primer used foramplification of the Cavia porcellus allergen 1a gene.

SEQ ID No. 31:

Nucleotide sequence of degenerate Cavp-MQ11 primer used foramplification of the Cavia porcellus allergen 1a gene.

SEQ ID No. 32:

Nucleotide sequence of degenerate Cavp-MQ12 primer used foramplification of the Cavia porcellus allergen 1a gene.

SEQ ID No. 33:

Nucleotide sequence of degenerate Cavp-MQ1rev primer used foramplification of the Cavia porcellus allergen 1a gene.

SEQ ID No. 34:

Nucleotide sequence of degenerate Cavp-MQ2rev primer used foramplification of the Cavia porcellus allergen 1a gene.

SEQ ID No. 35:

Nucleotide sequence of degenerate Cavp-MQ3rev primer used foramplification of the Cavia porcellus allergen 1a gene.

SEQ ID No. 36:

Nucleotide sequence of degenerate Cavp-MQ4rev primer used foramplification of the Cavia porcellus allergen 1a gene.

SEQ ID No. 37:

Nucleotide sequence of degenerate Cavp-MQ5rev primer used foramplification of the Cavia porcellus allergen 1a gene.

SEQ ID No. 38:

Nucleotide sequence of degenerate Cavp-MQ6rev primer used foramplification of the Cavia porcellus allergen 1a gene.

SEQ ID No. 39:

Nucleotide sequence of degenerate Cavp-MQ7rev primer used foramplification of the Cavia porcellus allergen 1a gene.

SEQ ID No. 40:

Nucleotide sequence of degenerate Cavp-MQ8rev primer used foramplification of the Cavia porcellus allergen 1a gene.

SEQ ID No. 41:

Nucleotide sequence of degenerate Cavp-11_for (3′RACE) primer used foramplification of the Cavia porcellus allergen 1a gene.

SEQ ID No. 42:

Nucleotide sequence of degenerate Cavp-b_rev (5′RACE) primer used foramplification of the Cavia porcellus allergen 1a gene.

1. An isolated nucleic acid molecule encoding a Caviidae allergencomprising a polynucleotide selected from the group consisting of: (a) apolynucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:5, SEQ IDNO:30R SEQ ID NO:19 or a fragment thereof; (b) a polynucleotide sequenceencoding a polypeptide as shown in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:4or SEQ ID NO:20 or a fragment thereof; (c) a polynucleotide sequencewhich has at least 80% identity to the polynucleotides as defined in (a)or (b) encoding a Caviidae allergen or a fragment thereof; (d) apolynucleotide sequence encoding a polypeptide which has at least 85%identity to the polypeptide as shown in SEQ ID NO:2, SEQ ID NO:6, SEQ IDNO:4 or SEQ ID NO:20 or a fragment thereof; (e) a polynucleotidesequence which hybridizes to the polynucleotide sequence of any one of(a) to (d) and whereby the coding strand encodes a Caviidae allergen ora fragment thereof; (f) a polynucleotide sequence encoding a polypeptideas encoded by the nucleotide sequence of any one of (a) to (e) whereinat least one amino acid is deleted, substituted, inserted or added andwhereby said polynucleotide encodes a Caviidae allergen or a fragmentthereof; and (g) a polynucleotide sequence being degenerate as a resultof the generic code to the nucleotide sequence as defined in any one of(a) to (f).
 2. A vector comprising the nucleic acid molecule of claim 1.3. A recombinant host cell comprising the nucleic acid molecule asdefined in claim
 1. 4. A method for producing a polypeptide, comprisingculturing the recombinant host cell of claim 3 under such conditionsthat the polypeptide is expressed, and recovering the polypeptide.
 5. Apolypeptide encoded by the nucleic acid molecule of claim 1, or afragment of said polypeptide, whereby said fragment is a Caviidaeallergen fragment.
 6. An antibody that specifically binds to thepolypeptide or fragment thereof as defined in claim
 5. 7. (canceled) 8.A pharmaceutical composition comprising the the polypeptide of claim 5.9. A method of hyposensibilization or medical intervention of anallergic reaction in a subject comprising administering thepharmaceutical composition of claim 8 to the subject.
 10. A method ofhyposensibilization or medical intervention of an allergic reaction in asubject comprising administering the polypeptide of claim 5 to thesubject.
 11. (canceled)
 12. A method for diagnosis of an allergicreaction in a subject or for the determination of a subject prone to anallergic reaction comprising exposing the subject to the polypeptide ofclaim 5 and determining an immunological reaction of the subject. 13.(canceled)
 14. An in vitro method for determining allergenicitycomprising the steps of: (a) exposing a sample to the polypeptideaccording to claim 5; and (b) determining immunological reaction or theformation of an immunological complex to the exposure of step (a). 15.The method of claim 14, wherein said sample is selected from the group,consisting of a biological sample, a medical sample or an environmentalsample.
 16. The method of claim 14, wherein said immunological reactioncomprises an increased titer of immunglobulines, an increased histaminerelease, leukotriene release or wherein said formation of animmunological complex is the formation of an antibody-antigen complexbetween the polypeptide and an antibody.
 17. The method of claim 16wherein an increased titer of immunglobulines and/or an increasedhistamine release is indicative for an immunological or allergicreaction.
 18. The method of claim 16 wherein said immunoglobuline isIgE.
 19. The method of claim 17, wherein said immunological or allergicreaction is a reaction to Caviidae.
 20. (canceled)
 21. (canceled)
 22. Amethod for identifying molecules which are capable of interacting withthe polypeptide of claim 5 comprising: (a) producing cells which expresssaid polypeptide; (b) contacting the polypeptide produced in step (a)with a test sample potentially containing said molecule; and (c)identifying among these molecules the molecules which are capable ofinteracting with said polypeptide.
 23. An in vitro method fordetermining allergenicity comprising the steps of: (a) exposing a sampleto the antibody of claim 6; and (b) determining immunological reactionor the formation of an immunological complex to the exposure of step(a).